Thrombin inhibitors

ABSTRACT

The invention relates to non-slow-binding thrombin inhibitors of the formula: A—B—C-Lys-D wherein A is H, 2-hydroxy-3-cyclohexylpropionyl-, R 1 , R 1 —O—CO—, R 1 —CO—, R 1 —SO 2 —, —(CHR 2 ) n COOR 3 , or an N-protecting group, wherein R 1  is selected from —(1-6C)alkylene-COOH, (1-12C)alkyl, (2-12C)alkenyl, (6-14C)aryl, (7-15C)aralkyl; and (8-16C)aralkenyl, the aryl group of which may be substituted with (1-6C)alkyl, (2-12C)alkoxy, hydroxy, or halogen; R 2  is H or has the same meaning as R 1 , R 3  is selected from H, (1-12C)alkyl, (2-12C)alkenyl, (6-14C)aryl, (7-15C)aralkyl and (8-16C)aralkenyl, the aryl group of which may be substituted with (1-6C)alkyl, (2-12C)alkoxy, hydroxy or halogen; n is an integer of 1 to 3; B is a bond, L-Asp or an ester derivative thereof, Leu, norLeu, —N(benzyl)-CH 2 —CO—, —N(2-indane)-CH 2 —CO—, D-1-Piq, D-3-Piq, D-Tiq, Atc or a D-amino acid having a hydrophobic aromatic side chain; C is Azt, Pro, Pec, norLeu(cyclo)Gly, an amino acid of one of the formulae —N[(3-8C)cycloalkyl]-CH 2 —CO— or —N(benzyl)-CH 2 —CO—, D is selected from COOH, tetrazole, oxazole, thiazole and benzothiazole, or A and C have the aforesaid meanings, B is D—(3-8C)cycloalkylalanine, and D is tetrazole, oxazole, thiazole or benzothiazole; or a prodrug thereof; or a pharmaceutically acceptable salt thereof; with the exception of the compound Me-D-Phe-Pro-Lys-COOH. The compounds can be used as antithrombotic agents.

The present invention relates to non-slow-binding thrombin inhibitors, aprocess for the preparation of said inhibitors, pharmaceuticalcompositions containing the same, and the use of these thrombininhibitors as antithrombotic agents.

BACKGROUND OF THE INVENTION

Much attention has been focused on inhibition of thrombin as potentialanticoagulants. Inhibitors of the enzyme thrombin, a key serine proteasewithin the blood coagulation cascade, have for some time been consideredas potential candidates for anticoagulant prophylaxis and therapy. Inparticular the multiple roles played by thrombin in its actions oncoagulation factors, circulating blood components, and the cells of thevessel wall makes it a particularly attractive target in a variety ofpathological states. Moreover, limitations associated with currentlyemployed anticoagulants, in particular the occurrence of bleedingcomplications, necessitate the search for more specifically-actingagents.

Many peptide(-like) serine protease inhibitors have been disclosed,amongst which are transition state inhibitors of thrombin. Many of theselatter compounds, however, are slow-binding inhibitors. The use ofslow-binding inhibitors of thrombin is open to criticism. In vivo,thrombin is constantly generated in plasma and thrombin inhibitorsprimarily function by slowing thrombin formation through inhibitingthrombin-mediated amplification steps. To slow down such anamplification cascade, a non-slow-binding inhibitor would be preferable.A larger dose of a slow-binding inhibitor would be needed to achieve thesame effect, with a correspondingly increased risk of side-effects.

Relevant thrombin inhibitors are disclosed by Brady et al., Bioorganic &Medicinal Chemistry, 3 (1995), 1063-78 wherein D-Phe-Pro-Arg-amide andD-Phe-Pro-Lys-X derivatives have been disclosed, X being a ketoester oramine. These compounds are disclosed to be slow-binding thrombininhibitors, and likewise these compounds are excluded from the presentinvention. In the search for non-slow-binding thrombin inhibitors Joneset al., J. Enzyme Inhibition, 9 (1995), 43-60 attempted to obtainimprovement by using D-Cha-Pro-Lys-COOH derivatives. However, althoughthese derivatives proved to be more potent thrombin inhibitors, theystill exhibit slow-binding properties.

In a recent attempt to obtain potent non-slow-binding thrombininhibitors Lewis et al., Thrombosis and Haemostasis, 74(4) (1995), 1107,prepared Me-D-Phe-Pro-Lys-X derivatives, X being carboxyamide orcarboxylic acid. These compounds, among which specifically disclosedMe-D-Phe-Pro-Lys-COOH, are classified as slow-binding inhibitors. Thiscompound therefore does not fulfill the requirements of the presentinvention and is excluded from protection.

A thrombin inhibitor with an alkyl-substituted lysine is disclosed inU.S. Pat. No. 5,523,308. In earlier references other Phe-Pro-Lyssequences are described, for example by Iwanowicz et al. in Bioorganic &Medicinal Chemistry Letters, 2 (1992), 1607-12, which disclosesD-Phe-Pro-Lys-X derivatives, X being i.a. a ketoester. Such compoundsmay also be described as slow-binding thrombin inhibitors.

Other types of peptides for inhibition of different serine proteases arealso disclosed. Tsutsumi et al. in J. Med. Chem., 37 (1994), 3492-3502described peptide-like compounds having thiazole and benzothiazoleC-terminal ends. It was found that such thiazole derivatives are 300times more potent than the corresponding thiophene analogues. It wasfurther posited that C-terminal heterocyclic groups would provide acritical hydrogen-bond interaction with the histamine of the proteaseprolyl endopeptidase. Although it was further suggested that thisfeature may well be capable of extension to other serine proteases,thrombin proteases were not specifically mentioned. The mechanisticexplanation of Tsutsumi was later challenged by Edwards et al. in J.Med. Chem., 38 (1995), 76-85, but also these authors found that elastaseinhibitors of the type D-Phe-Val-Pro-Val-X, X being thiazole andbenzothiazole, are non-slow-binding inhibitors of the relevant serineprotease. These authors suggest the development of peptidylα-ketoheterocycles as inhibitors of other serine proteases as well.

SUMMARY OF THE INVENTION

The present invention relates to the surprising finding that theteachings of Edwards, Tsutsumi and others can also be applied tothrombin inhibitors. The application of the C-terminal heterocycles tothe compounds as disclosed by Lewis, Jones and Brady provide potentthrombin inhibitors having non-slow-binding properties to thrombin.Moreover, many of these compounds show improved biological half-lifesand oral bioavailability.

The invention therefore relates to non-slow-binding thrombin inhibitorsof the formula:

A—B—C-Lys-D

wherein

A is H, 2-hydroxy-3-cyclohexyl-propionyl-, R₁, R₁—O—CC—, R₁—CO— ,R₁—SO₂—, —(CHR₂)_(n)COOR₃, or an N-protecting group, wherein

R₁ is selected from —(1-6C)alkylene-COOH, (1-12C)alkyl, (2-12C)alkenyl,(6-14C)aryl, (7-15C)aralkyl and (8-16C)aralkenyl, the aryl group ofwhich may be substituted with (1-6C)alkyl, (2-12C)alkoxy, hydroxy, orhalogen;

R₂ is H or has the same meaning as R₁;

R₃ is selected from H, (1-12C)alkyl, (2-12C)alkenyl, (6-14C)aryl,(7-15C)aralkyl and (8-16C)aralkenyl, the aryl group of which may besubstituted with (16C)alkyl, (2-12C)alkoxy, hydroxy, or halogen;

n is an integer of 1 to 3;

B is a bond, L-Asp or an ester derivative thereof, Leu, norLeu,—N(benzyl)—CH₂—CO—, —N(2-indane)—CH₂—CO—, D-1-Piq, D-3-Piq, D-Tiq, Atcor a D-amino acid having a hydrophobic aromatic side chain;

C is Azt, Pro, Pec, norLeu(cyclo)Gly, or an amino acid of one of theformulae —N[(3-8C)cycloallkyl]-CH₂—CO— or —N(benzyl)-CH₂—CO—;

D is selected from COOH, tetrazole, oxazole, thiazole and benzothiazole;

or A and C have the aforesaid meanings, B is D—(3-8C)cycloalkylalanine,and D is tetrazole, oxazole, thiazole or benzothiazole;

or a prodrug thereof,

or a pharmaceutically acceptable salt thereof;

with the exception of the compound Me-D-Phe-Pro-Lys-COOH.

The compounds of the present invention are useful for treating andpreventing thrombin-mediated and thrombin-associated diseases. Thisincludes a number of thrombotic and prothrombotic states in which thecoagulation cascade is activated which include, but are not limited to,deep vein thrombosis, pulmonary embolism, thrombophlebitis, arterialocclusion from thrombosis or embolism, arterial reocclusion during orafter angioplasty or thrombolysis, restenosis following arterial injuryor invasive cardiological procedures, postoperative venous thrombosis orembolism, acute or chronic atherosclerosis, stroke, myocardialinfarction, cancer and metastasis, and neurodegenerative diseases. Thecompounds of the invention may also be used as anticoagulants inextracorporeal blood circuits, as necessary in dialysis and surgery. Thecompounds of the invention may also be used as in vitro anticoagulants.

DETAILED DESCRIPTION OF THE INVENTION

Preferred compounds according to this invention are the compoundswherein D is COOH. In addition, preferably A is H, (1-12C)alkyl,—CO—(7-15C)aralkyl, —SO₂—(1-12C)alkyl, —SO₂—(6-14C)aryl, or—SO₂(7-15C)aralkyl; B is a bond, L-Asp, norLeu, D-1-Piq, or D-Phe; and Cis Pro, norLeu(cyclo)Gly, or —N-cyclopentyl—CH₂—CO—. More preferred arethe non-slow-binding thrombin inhibitors wherein A is —SO₂-benzyl, B isa bond, and C is norLeu(cyclo)Gly, or wherein A is —SO₂-ethyl, B isD-Phe, and C is Pro; or wherein A is hydrogen, B is D-1-Piq, and C isPro.

Other preferred compounds according to the invention are those wherein Dis oxazole or thiazole. Further, preferably A is H, (1-12C)alkyl,2-hydroxy-3-cyclohexyl-propionyl-, —CO—(CH₂)_(n)COOH,—CO—(7-15C)aralkyl, —SO₂—(6-14C)aryl, —SO₂—(7-15C)aralkyl,—SO₂—(1-12C)alkyl, —(CHR₂)_(n)COOR₃, R₂ being H or (1-12Calkyl) and R₃being H, (1-12C)alkyl or benzyl; and C is Pro, norLeu(cyclo)Gly, or—N[(3-8C)cycloalkyl]-CH₂—CO—. Particularly preferred are thenon-slow-binding thrombin inhibitor wherein A is —(CH₂)_(n)COOR₃, R₃being H, (1-12C)alkyl or benzyl; B is D—(3-8C)cycloalkylalanine, orD-Phe optionally monosubstituted with alkoxy or halogen; and C is Pro.The most preferred compounds of the invention are compounds wherein D isthiazole. Specifically preferred is the non-slow-binding inhibitorHOOC—CH₂D-Cha-Pro-Lys-(2-thiazolyl).

The N-protecting group as defined in the definition of moiety A is anyN-protecting group as used in peptides. Suitable N-protecting groups canbe found in T. W. Green and P. G. M. Wuts: Protective Groups in OrganicSynthesis, Second Edition (Wiley, N.Y., 1991) and in The Peptides,Analysis, Synthesis, Biology, Vol. 3 E. Gross and J. Meienhofer, Eds.,(Academic Press, New York, 1981).

Alkyl, as used herein, is a branched or unbranched alkyl group having 1to 12 carbon atoms, such as methyl, ethyl, isopentyl, dodecyl, and thelike.

The term (1-6C)alkylene means a branched or unbranched alkylene grouphaving 1 to 6 carbon atoms, such as —(CH₂)_(m)— and m is 1 to 6,—CH(CH₃)—, —CH(CH₃)—(CH₂)—, etc. The preferred alkylene group ismethylene.

Alkenyl is a branched or unbranched unsaturated alkenyl group having 2to 12 carbon atoms. Examples are ethenyl, propenyl, allyl, and the like.

Aralkyl and aralkenyl groups are alkyl and alkenyl groups respectively,substituted by one or more aryl groups, the total number of carbon atomsbeing 7 to 15 and 8 to 16, respectively. Preferred aralkyl groups aree.g. of the formulae —(CH₂)_(p)—CH—(C6H₅)₂, p being 1 or 2, or—CH₂)_(q)—C₆H₅ optionally substituted with halogen, q being 1,2 or 3.

Aryl in above-mentioned definition and in the definition of aryl, asused in the compound of the invention, is an aromatic moiety of 6 to 14carbon atoms. The aryl group may further contain one or more heteroatoms, such as N, S, or O. Examples of aryl groups are phenyl, naphthyl,(iso)quinolyl, indanyl, and the like. Most preferred is the phenylgroup. The aryl group may be substituted with on or more alkyl groups,preferably methyl, alkoxy groups, preferably methoxy, hydroxy, orhalogen. The term halogen means fluorine, chlorine, bromine or iodine.Chlorine is the preferred halogen.

The terms D-1-Piq and D-3-Piq mean 1- and 3-carboxyperhydroisoquinoline,respectively. The term Tiq means1,2,3,4-tetrahydroisoquinoline-carboxylic acid. Atc is2-aminotetralin-2-carboxylic acid. The terms Azt and Pec mean2-azetidine carboxylic acid and pipecolinic acid, repectively.

The term norLeu(cyclo)Gly means a structural fragment of the formula

The term hydrophobic aromatic side chain means a (1-12C)alkylsubstituted with one or more (6-14C)aryl groups (which may contain aheteroatom, e.g. nitrogen) such as phenyl, pyridinyl, naphthyl,tetrahydronaphthyl, and the like, which hydrophobic side chain may besubstituted with hydrophobic substituents such as halogen (preferablychlorine), trifluoromethyl, lower alkyl (for instance methyl or ethyl),lower alkoxy (for instance methoxy), phenyloxy, benzyloxy, and the like.

The term (3-8C)cycloalkyl means cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl, cycloheptyl or cyclooctyl.

Tetrazole, oxazole, thiazole and benzothiazole have the followingformulae, respectively:

The invention also includes prodrugs of the compounds of the generalformula, which after administration are metabolized into the activecompounds. Suitable prodrugs are for example N-alkoxycarbonyl protected(preferably N-ethoxycarbonyl) derivatives of the general formula.

As used herein the term pharmaceutically acceptable salt refers to saltsthat retain the desired biological activity of the parent compound andpreferably do not impart any undesired toxic effects. Examples of suchsalts are acid addition salts formed with inorganic acids, for examplehydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acidnitric acid, and the like. Salts may also be formed with organic acidssuch as, for example, acetic acid, oxalic acid, tartaric acid, succinicacid, maleic acid, fumaric acid, gluconic acid, citric acid, malic acid,ascorbic acid, benzoic acid, tannic acid, pamoic acid, alginic acid,polyglutamic acid, and the like. Salts may be formed with polyvalentmetal cations such as zinc, calcium, bismuth, barium, magnesium,aluminum, copper, cobalt, nickel and the like, or with an organic cationformed from N,N′-dibenzylethylenediamine or ethylenediamine, orcombinations thereof (e.g. a zinc tannate salt).

The compounds of this invention possess one or more chiral carbon atoms,and may therefore be obtained as a pure enantiomer, or as a mixture ofenantiomers, or as a mixture containing diastereomers. Methods forobtaining the pure enantiomers are well known in the art, e.g.crystallization of salts which are obtained from optically active acidsand the racemic mixture, or chromatography using chiral columns. Fordiastereomers straight phase or reversed phase columns may be used.

The invention further includes a process for preparing a compound of theformula, the process including coupling suitably protected amino acidsor amino acid analogs, followed by removing the protecting groups.

The compounds according to the general formula may be prepared in amanner conventional for such compounds. To that end, suitably Naprotected (and side-chain protected if reactive side-chains are present)amino acid derivatives or peptides are activated and coupled to suitablycarboxyl protected amino acid or peptide derivatives either in solutionor on a solid support. Protection of the α-amino functions generallytakes place by urethane functions such as the acid-labiletert.-butyloxycarbonyl group (Boc), benzyloxycarbonyl (Z) group andsubstituted analogs or the base-labile 9-fluorenyl-methyloxycarbonyl(Fmoc). group. The Z group can also be removed by catalytichydrogenation. Other suitable protecting groups include the Nps, Bmv,Bpoc, Aloc, MSC, etc. A good overview of amino protecting groups isgiven is given in The Peptides, Analysis, Synthesis, Biology, Vol. 3 E.Gross and J. Meienhofer, Eds., (Academic Press, New York, 1981).Protection of carboxyl groups can take place by ester formation e.g.base-labile esters like methyl or ethyl, acid labile esters like tert.butyl, or hydrogenolytically-labile esters like benzyl. Protection ofside-chain functions like those of lysine can take place using theaforementioned groups. Activation of the carboxyl group of the suitablyprotected amino acids or peptides can take place by the azide, mixedanhydride, active ester, or carbodiimide method, especially with theaddition of catalytic and racemization-suppressing compounds like1-hydroxybenzotriazole, N-hydroxysuccinimide,3-hydroxy4-oxo-3,4-dihydro-1,2,3,-benzotriazine,N-hydroxy-5-norbornene-2,3-dicarboximide. Also the anhydrides ofphosphorus based acids can be used. See, e.g. The Peptides, Analysis,Synthesis, Biology, supra and Pure and Applied Chem. 59(3), 331-344(1987).

It is also possible to prepare the compounds by the solid phase methodof Merrifield. Different solid supports and different strategies areknown see, e.g. Barany and Merrifield in The Peptides, Analysis,Synthesis, Biology, Vol. 2, E. Gross and J. Meienhofer, Eds., (Acad.Press, N.Y., 1980), Kneib-Cordonier and Mullen Int. J. Peptide ProteinRes., 30, 705-739 (1987) and Fields and Noble Int. J. Peptide ProteinRes., 35, 161-214 (1990).

Removal of the protecting groups, and, in the case of solid phasepeptide synthesis, the cleavage from the solid support, can take placein different ways, depending on the nature of those protecting groupsand the type of linker to the solid support. Usually deprotection takesplace under acidic conditions and in the presence of scavengers. See,e.g. volumes 3, 5 and 9 of the series on The Peptides Analysis,Synthesis, Biology, supra.

Another possibility is the application of enzymes in synthesis of suchcompounds; for reviews see e.g. H. D. Jakubke in The Peptides, Analysis,Synthesis Biology, Vol. 9, S. Udenfriend and J. Meienhofer, Eds., (Acad.Press, N.Y., 1987).

However made, the compounds are useful for the manufacture ofmedicaments which have use in treating disease states involvingundesired blood coagulation. In such a case the particular compoundsynthesized will typically be associated with a pharmaceutical carrier.Pharmaceutical carriers vary from things as relatively simple assterilized water for injection to things as relatively complicated asmicrospheres and biodegradable implants.

As medicaments, the compounds are preferably administered orally,subcutaneously, topically, intranasally, intra-venously, intramuscularlyor locally (e.g. via an implant). Depot administration is also possible.

The exact dose and regimen for administration of these compounds andcompositions will necessarily be dependent upon the needs of theindividual subject to whom the medicament is being administered, thedegree of affliction or need, and of course, the judgment of the medicalpractitioner. In general parenteral administration requires lowerdosages than other methods of administration which are more dependentupon absorption. Illustratively however, the dosages are in the range of0.001-100 mg per kg body weight, preferably 0.01-10 mg per kg bodyweight.

The medicament manufactured with the compounds may also be used asadjuvant in acute anticoagulant therapy. In such a case, the medicamentis administered with other compounds useful in treating such diseasestates.

The compounds may also be used with implantable pharmaceutical devicessuch as those described in U.S. Pat. No. 4,767,628, the contents ofwhich are incorporated by this reference. Then the device will containsufficient amounts of compound to slowly release the compound (e.g. formore than a month).

Methods of making medicaments which can be adapted to contain thecompound for enteral or parenteral administration are described in thestandard reference, Gennaro et al., Remington's Pharmaceutical Sciences,(18th ed., Mack Publishing Company, 1990, see especially Part 8:Pharmaceutical Preparations and Their Manufacture), pages 1519 through1580. Mixed with pharmaceutically suitable auxiliaries, the compoundsmay be compressed into solid dosage units, such as pills, tablets, or beprocessed into capsules or suppositories. By means of pharmaceuticallysuitable liquids the compounds can also be applied in the form of asolution, suspension, emulsion, e.g. for use as an injectionpreparation, or as a spray, e.g. for use as a nasal spray.

For making dosage units, e.g. tablets, the use of conventional additivessuch as fillers, colorants, polymeric binders and the like iscontemplated. In general any pharmaceutically acceptable additive whichdoes not interfere with the function of the active compounds can beused. Suitable carriers with which the compositions can be administeredinclude lactose, starch, cellulose derivatives and the like, or mixturesthereof, used in suitable amounts.

The invention is further explained by reference to the followingillustrative Examples.

EXAMPLE 1 3,3-Diphenylpropionyl-Pro-Lys-(2-thiazolyl)3,3-Diphenylpropionyl-prolyl-methylester

To a cold solution (0° C.) of 3,3-diphenylpropionic acid (5.0 g) inethyl acetate (100 ml) were successively added DCCI(1,3-dicyclohexylcarbodiimide; 5.03 g), HOBt (1-hydroxy-benzotriazolehydrate; 3.28 g), H-Pro-OMe.HCl (3.66 g) and triethylamine (3.1 ml). Thereaction mixture was stirred at 0° C. for 1 h and then kept at roomtemperature overnight. The reaction mixture was cooled to −20° C. andDCU (1,3-dicyclohexylurea) was removed by filtration. The filtrate waswashed successively with 5% sodium hydrogencarbonate, water, 5%potassium hydrogensulfate and saturated aqueous sodium chloride; driedover sodium sulfate and concentrated in vacuo. The residue was purifiedby chromatography on silica (eluent: dichloromethane/ethyl acetate; 9/1v/v) to yield 5.68 g of 3,3-diphenylpropionyl-prolylmethylester as acrystalline powder. TLC: R_(f)=0.75, silica gel, dichloromethane/ethylacetate=7/3 v/v.

3,3-Diphenylpropionyl-prolyl-OH

3,3-Diphenylpropionyl-prolyl-methylester (5.6 g) dissolved indioxane/water; 7/3 v/v (60 ml) was treated with a 4 M sodium hydroxidesolution (6.2 ml) portionwise over 30 min at room temperature, keepingthe pH at 10-10.5. After 30 min the reaction mixture was diluted withwater (60 ml), a 4 M hydrochloric acid solution was added until pH 2.0and the water layer was extracted with ethyl acetate. The combinedorganic phases were washed with water, saturated aqueous sodium chlorideand dried over sodium sulfate and the solvent was removed by evaporationyielding 3,3-diphenylpropionyl-prolyl-OH as a syrup (5.18 g). TLC:R_(f)=0.65, silica gel, EPAW (ethyl acetate/pyridine/acetic acid/water)63/20/6/11 v/v/v/v.

Boc-Lys(Cbz)-NMeOMe

Boc-Lys(Cbz)-OH.DCHA (10 g) was suspended in dichloromethane (200 ml).The suspension was washed with cold 0.1 N hydrochloric acid solutiontwice. 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluroniumtetrafluoroborate (6.0 g) and O,N-dimethyl-hydroxylamine hydrochloricacid (1.82 g) were added to the resulting organic phase and the pH wasadjusted to pH 8 by adding triethylamine. The reaction mixture wasstirred for 1 h at room temperature. The mixture was washed successivelywith cold 2N hydrochloric acid solution, water, 5% sodiumhydrogencarbonate, and water. The organic layer was dried over sodiumsulfate, filtered and evaporated. The residue was purified bychromatography on silica (eluent: dichloromethane/methanol; 5/5 v/v) toyield Boc-Lys(Cbz)-NMeOMe (7.2 g). TLC: R_(f)=0.55, silica gel,dichloromethane/ methanol 95/5 v/v.

Boc-Lys(Cbz)-(2-thiazolyl)

To a cold (−78° C.), stirred solution of n-butyllithium (63.9 mmol) indiethyl ether (58 ml), was added dropwise a solution of 2-bromothiazole(10.5 g) in diethyl ether (30 ml). The solution was stirred at −78° C.for 30 min, after which a solution of Boc-Lys(Cbz)-NMeOMe (8.2 g) in dryTHF (tetrahydrofuran; 75 ml) was added slowly. The mixture was stirredat −78° C. for 1 h, then 5% aqueous sodium hydrogencarbonate was added.The mixture was allowed to warm to room temperature and the layers wereseparated. The aqueous layer was extracted with diethyl ether. Thecombined organic layers were washed with water, dried over sodiumsulfate, filtered and evaporated. The residue was purified bychromatography on silica (eluent: ethyl acetate/heptane; 3/1 v/v) toyield Boc-Lys(Cbz)-(2-thiazolyl) (8.6 g). TLC: R_(f)=0.77, silica gel,ethyl acetate/heptane=3/1 v/v.

H-Lys(Cbz)-(2-thiazolyl).TFA

Boc-Lys(Cbz)-(2-thiazolyl) (500 mg) was dissolved in 50%TFA(trifluoroacetic acid)/dichloromethane (5 ml) and stirred for 1 h atroom temperature. The crude H-Lys(Cbz)-(2-thiazolyl).TFA was isolated inquantitative yield after removal of the solvent by evaporation, and usedimmediately in the next step. TLC: R_(f)=0.25, silica gel, EPAW=63/20/6/11 v/v/v/v.

3,3-Diphenylpropionyl-Pro-Lys(Cbz)-(2-thiazolyl)

To a cold (0° C.) solution of 3,3-diphenylpropionyl-prolyl-OH (385 mg)in dimethyl formamide (5 ml) were successively added DCCI (270 mg), HOBt(176 mg), H-Lys(Cbz)-(2-thiazolyl).TFA (515 mg) and N-ethylmorpholine(0.28 ml). The reaction mixture was stirred at 0° C. for 1 h and thenkept overnight at room temperature. The mixture was cooled to −20° C.and DCU was removed by filtration. The filtrate was evaporated todryness. The residue was dissolved in ethyl acetate and washedsuccessively with 5% aqueous sodium hydrogen-carbonate, water, 5%aqueous potassium hydrogensulfate and saturated aqueous sodium chloride,dried over sodium sulfate and concentrated in vacuo. The residue waspurified by chromatography on silica (eluent: ethyl acetate/heptane; 4/1v/v) to yield 3,3-diphenylpropionyl-Pro-Lys(Cbz)-(2-thiazolyl) (332 mg).TLC: R_(f)=0.40, silica gel, ethyl acetate/heptane; 3/1 v/v.

3,3-Diphenylpropionyl-Pro-Lys-(2-thiazolyl)

3,3-Diphenylpropionyl-Pro-Lys(Cbz)-(2-thiazolyl) (320 mg) was treatedwith TFA/thioanisole 10/1 v/v (3.3 ml) for 3 h at room temperature. Thereaction mixture was concentrated in vacuo and the residue was dissolvedin water. The aqueous phase was washed extensively with diethyl ether.

The water layer, containing 3,3-diphenylpropionyl-Pro-Lys-(2-thiazolyl)was charged directly onto a preparative HPLC Supelcosil LC-18-DB columnusing a gradient elution system of 20% A/60% B/20% C to 20% A/80% C over45 min, at a flow rate of 20 ml/min. (A: 0.5 M sodium phosphate bufferpH 2.1, B: water, C: acetonitrile/water; 3/2 v/v).

Yield: 47 mg of 3,3-diphenylpropionyl-Pro-Lys-(2-thiazolyl). TLC:R_(f)=0.57, silica gel, EPAW; 63/20/6/11 v/v/v/v. R_(t) (LC): 32.9 min20% A/60% B/20% C to 20% A/0% B/80/% C in 40 min.

EXAMPLE 2

In a similar manner as described in Example 1 were prepared:

(a). H—D-Phe-ProLys-(2-thiazolyl)

R_(t) (LC): 25.67 min; 20% A/80% B/0% C to 20% A/20% B/60% C in 40 min.

(b). H—D-1-Tiq-Pro-Lys-(2-thiazolyl)

R_(t) (LC): 23.40 min; 20% A/80% B/0% C to 20% A/20% B/60% C in 40 min.(Tiq=tetrahydroisoquinolin)

(c). H—D—(p—Cl)-Phe-Pro-Lys-(2-thiazolyl)

R_(t) (LC): 30.47 min; 20% A/80% B/0% C to 20% A/20% B/60% C in 40 min.

(d). Indaneglycyl-(N-cyclopropyl)-Gly-Lys-(2-thiazolyl)

R_(t) (LC): 27.88 min; 20% A/80% B/0% C to 20% A/20% B/60% C in 40 min.

(e). H—D-Phe-(N-cyclopentyl)-Gly-Lys-(2-thiazolyl)

R_(t) (LC): 31.07 min; 20% A/80% B/0% C to 20% A/20% B/60% C in 40 min.

(f). Acetyl-D-Phe-(N-cyclopropyl)-Gly-Lys-(2-thiazolyl)

R_(t) (LC): 33.73 min; 20% A/80% B/0% C to 20% A/20% B/60% C in 40 min.

(g). H—D-Cha-Pro-Lys-(2-thiazolyl)

R_(t) (LC): 30.59 min; 20% A/80% B/0% C to 20% A/20% B/60% C in 40 min.(Cha=cycdohexylalanine)

(h). H—D-Phe-(N-cyclopropyl)Gly-Lys-(2-thiazolyl)

R_(t) (LC): 5.1 min isocratic; 55/45 MeOH/25 mM phosphate pH=7.

(i). 3,3-Diphenylpropionyl-(N-cyclopropyl)-Gly-Lys-(2-thiazolyl)

R_(t) (LC): 8.1 min isocratic; 75/25 MeOH/25 mM phosphate pH=7.

(j). H—D-Phe-(N-cyclobutyl)-Gly-Lys-(2-thiazolyl)

R_(t) (LC): 30.59 min; 20% A/80% B/0% C to 20% A/20% B/60% C in 40 min.

(k). H-Atc-Pro-Lys-(2-thiazolyl)

R_(t) (LC): 27.79+28.04 min; 20% A/80% B/0% C to 20% A/20% B/60% C in 40min. (Atc=2-aminotetralin-2-carboxylic acid)

(l). H—D-Phe-(N-benzyl)-Gly-Lys-(2-thiazolyl)

R_(t) (LC): 16.99 min; 20% A/60% B/20% C to 20% A/0% B/80% C in 40 min.

(m). H—D—Cha-(N-cyclopropyl)-Gly-Lys-(2-thiazolyl)

R_(t) (LC): 30.84 min; 20% A/80% B/0% C to 20% A/20% B/60% C in 40 min.

(n). p-chloro-3-phenylpropionyl-(N-cyclopentyl)-Gly-Lys-(2-thiazolyl)

R_(t) (LC): 36.15 min; 20% A/60% B/20% C to 20% A/0% B/80% C in 40 min.

(o). (N-benzyl)-Gly-(N-cyclopentyl)-Gly-Lys-(2-thiazolyl)

R_(t) (LC): 28.14 min; 20% A/80%B/0% C to 20%A/80% C in 40 min.

EXAMPLE 3 HOOC—CH₂—D-Cha-(N-cyclopentyl)-Gly-Lys-(2-thiazolyl)N-Cyclopentyl-Gly-OMe

To a solution of 23.2 g of H-Gly-OMe.HCl in 200 ml of methanol was added15.6 g of cyclopentanone. The mixture was stirred for 15 min. and 7 g ofsodium cyanoborohydride was added. The pH was adjusted to 6. Thereaction mixture was stirred for 16 h at room temperature. To completethe reaction 1 g of cyclopentanone was added and stirring was continued.The reaction was monitored on TLC. When all the starting material haddissappeared, the mixture was acidified to pH 2 and stirred for 30 min.The solvent was removed and the residue diluted with water. The solutionwas washed with ether, the pH adjusted to 12 with 6N sodium hydroxideand extracted with dichloromethane. The combined organic layers werewashed with saturated sodium chloride solution, dried on sodium sulfateand evaporated in vacuo to yield 16 g of an oil.

R_(f)=0.46 in ethyl acetate/pyridine/acetic acid/water 63/20/6/11v/v/v/v on silica.

N-(t-butyloxycarbonyl-methyl)-D-Cha-OMe

To a stirred solution of 26 g of H—D-Cha-OMe.HCl in 300 ml ofacetonitrile was added 17 g of t-butyl-bromo acetate. The pH of thereaction was adjusted to 8.5 with diisopropylethylamine. The mixture wasstirred for 16 h at room temperature and evaporated in vacuo. Theresidue was dissolved in dichloromethane and the solution was washedwith water, dried on sodium sulfate and evaporated in vacuo.Chromatography over silica gel in hexane:ethyl acetate 9:1 v/v gave 20 gof the title product.

R_(f)=0.46 in ethyl acetate/pyridine/acetic acid/water 15.75/5/1.5/2.75v/v/v/v on silica.

N,N-Boc,(t-butyloxycarbonyl-methyl)-D-Cha-OMe

The pH of a solution of 20 g of N-(t-butyloxy-methyl)-D-Cha-OMe and 17 gof di-t-butyl dicarbonate was adjusted to pH 8.5 withdiisopropylethylamine. The mixture was stirred for 16 h at roomtemperature. The solvent was removed in vacuo. To the residue was addeddichloromethane and water. The organic layer was separated, washed withcold 1N hydrogen chloride, water, 5% sodium hydrogencarbonate and water.The organic layer was dried on sodium sulfate and the filtrate wasevaporated to an amorphous solid with a yield of 28 g.

R_(f)=0.60 in ethyl acetate/pyridine/acetic acid/water 252/20/6/11v/v/v/v on silica.

N,N-Boc,(t-butyloxycarbonyl-methyl)-D-Cha-OH

A solution of 28 g of N,N-Boc,(t-butylcarbonyl-methyl)D-Cha-OMe in 420ml of dioxane:water 9/1 was treated with sufficient 1N sodium hydroxideto keep the pH at 13 for 90 min. at room temperature. Afteracidification, the mixture was poured into water and extracted withdichloromethane. The organic layer was washed with water and dried onsodium sulfate. The filtrate was evaporated and yielded 24 g of thetitle compound.

R_(f)=0.23 in dichloromethane/methanol 9/1 v/v on silica.

N,N-Boc,(t-butyloxycarbonyl-methyl)-D-Cha-(N-cyclopentyl)-Gly-OMe

To a solution of 24 g of N,N-Boc,(t-butyloxycarbonyl-methyl)-D-Cha-OH in300 ml of N,N-dimethylformamide was added 10.2 g N-cyclopentyl-Gly-OMeand 21.2 g of 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluroniumtetrafluoroborate (TBTU).

The pH of the mixture was adjusted to 8.5. The mixture was stirredovernight at room temperature and concentrated by evaporation. Water andethyl acetate were added to the residue. The organic layer was separatedand washed with 1N hydrogen chloride, water, 5% sodium hydrogencarbonateand water and dried over sodium sulfate. The filtrate was evaporated andthe residue was chromatographed on silica gel in hexane:ethyl acetate8:2 as eluent. The fractions containing the title product are pooled andevaporated. Yield: 17 g. Rf=0.57 in hexane/ethyl acetate 7/3 v/v onsilica.

N,N-Boc,(t-butyloxycarbonyl-methyl)-D-Cha-(N-cyclopentyl)-Gly-OH

Using the same procedure as forN,N-Boc,(t-butyloxycarbonyl-methyl)-D-Cha-OH, 17 g ofN,N-Boc,(t-butyloxycarbonyl-methyl)-D-Cha-(N-cyclopentyl)-Gly-OMe issaponified and yielded 15 g of an amorphous solid. Chromatography oversilica gel with dichloromethane/methanol 95/5 v/v as eluent gave 13 g ofthe title compound.

Rf=0.30 in methylene chloride/methanol 9/1 v/v on silica.

HOOC—CH₂—D-Cha-(N-cyclopentyl)-Gly-Lys-(2-thiazolyl)

Using N,N-Boc,(t-butyloxycarbonyl-methyl)-D-Cha-(N-cyclopentyl)-Gly-OHin similar procedures as described for example 1, this compound wasprepared.

Rt (LC): 23.57 min; 20% A, 60% B, 20% C to 20% A, 80% C in 40 min.

(a). In a similar manner as described above using Proline.HCl and HONSu(as in example 11) HOOC—CH₂—D-Cha-Pro-Lys-(2-thiazolyl) was prepared. Rt(LC): 31.44 min; 20% A, 80% B, 0% C to 20% A, 20% B, 60% C in 40 min.

EXAMPLE 4 3-Phenylpropionyl-Pro-LysΨ[COCO]—OH Boc-Lys(Cbz)-OMe

Boc-Lys(Cbz)-OH (25 g) was dissolved in dichloromethane/methanol=9/1 v/v(500 ml). TBTU (21.1 g) was added and the solution adjusted to pH 8 byaddition of triethylamine. The reaction mixture was stirred for 1 h atroom temperature. The mixture was washed successively with cold 2Nhydrogen chloride solution, water, 5% sodium hydrogencarbonate, andwater. The organic layer was dried over sodium sulfate, filtered andevaporated. The residue was purified by chromatography on silica(eluent: ethyl acetate/heptane =1/4 v/v) to yield Boc-Lys(Cbz)-OMe (26.7g). TLC: R_(f)=0.79, silica gel, ethyl acetate/heptane=3/1 v/v.

Boc-Lys(Cbz)Ψ[cyanoacetate]

To a cold (−78° C.) solution of Boc-Lys(Cbz)-OMe (20 g) in drydichloromethane (600 ml) was added dropwise diisobutyl aluminumhydride(127 ml of 1M solution in hexane) at a rate to keep the reactiontemperature below −70° C. The resulting solution was stirred at −78° C.for 30 min. A 5% citric acid solution (500 ml) was added to the reactionmixture. The two layer mixture was stirred at room temperature for 10min, the layers were separated and the aqueous layer was extracted twicewith dichloromethane. The combined dichloromethane layers were washedwith water and dried over sodium sulfate and filtered. The solution wasplaced under nitrogen and cooled on a icewater-bath. A solution ofsodium cyanide (24.85 g) and benzyltriethyl ammonium chloride (2.89 g)in water (500 ml) was added. Under vigorous stirring acetic anhydridewas added portionwise (6×6 ml) over a period of 30 min. The organiclayer was separated and the aqueous layer was extracted twice withdichloromethane. The combined dichloromethane layers were washed withwater, dried over sodium sulfate, filtered and evaporated in vacuo. Theresidue was purified by chromatography on silica (eluent:dichloromethane/ethyl acetate=9/1 v/v) to yieldBoc-Lys(Cbz)Ψ[cyanoacetate] (17.2 g.). TLC: R_(f)=0.60, silica gel,dichloromethane/ethyl acetate=7/3 v/v.

Boc-Lys(Cbz)Tr[CHOHCO]—OMe

A solution of Boc-Lys(Cbz)Ψ[cyanoacetate] (17.2 g.) in diethylether/methanol=3/1 v/v (500 ml) was cooled to −20° C. under nitrogen,and 54.7 g of gaseous hydrochloric acid was introduced keeping thetemperature below −5° C. The reaction mixture was kept at 4° C.overnight. Water (85 ml) was added dropwise to the reaction mixturekeeping the temperature below 5° C. After stirring for 4 h at roomtemperature the organic layer was separated and washed with water. Theaqueous layer was saturated with sodium chloride and extracted withsec-butanoldichloromethane=3/2 v/v. The organic phase was washed withbrine, dried over sodium sulfate, filtered and evaporated in vacuo togive 17.4 g of the crude amine.

The residue was taken up in dimethyl formamide (DMF, 200 ml),bis-(tert-butyl)anhydride (8.7 g) was added and triethylamine until pH8. The reaction mixture was stirred at room temperature overnight. Thesolvent was removed by evaporation at reduced pressure. The residue wasdissolved in ethyl acetate, washed with water and brine successively,dried over sodium sulfate, filtered and evaporated in vacuo. The residuewas purified by chromatography on silica (eluent: ethylacetate/heptane=1/1 v/v) to yield Boc-Lys(Cbz)Ψ[CHOHCO]—OMe (6.22 g).TLC: R_(f)=0.75, silica gel, ethyl acetate/heptane=3/1 v/v.

3-Phenylpropionyl-Pro-Lys(Cbz)Ψ[CHOHCO]—OMe

Boc-Lys(Cbz)Ψ[CHOHCO]—OMe (60 mg.) was dissolved in 50% trifluoroaceticacid/dichloromethane (6 ml) and stirred for 1 h at room temperature. Thecrude amine was isolated in quantitative yield after removal of thesolvent by evaporation, and used immediately to prepare3-phenylpropionyl-Pro-Lys(Cbz)Ψ[CHOHCO]—OMe.

3-Phenylpropionyl-Pro-OH was dissolved in dry DMF (5 ml). After additionof triethylamine (196 ml), the reaction mixture was placed undernitrogen and cooled to —15° C. Isobutylchloroformate (183 ml) wassubsequently added and the mixture was allowed to stir for 15 min at−15° C. The crude amine was dissolved in dry DMF (5 ml), neutralisedusing triethylamine and added dropwise to the cold mixed anhydridesolution. The reaction stirred for 1 h at −15° C. and then kept at 0° C.overnight. The mixture was evaporated to dryness. The residue wasdissolved in ethyl acetate and successively wsahed with water, 5% sodiumhydrogencarbonate and brine, dried over sodium sulfate and concentratedin vacuo. The residue was purified by chromatography on silica (eluent:dichloromethane/methanol=95/5 v/v) to yield3-phenylpropionyl-Pro-Lys(Cbz)Ψ[CHOHCO]—OMe (246 mg). TLC: R_(f)=0.92,silica gel, EPAW=63/20/6/11 v/v/v/v.

3-Phenylpropionyl-Pro-LysΨ[CHOHCO]—OMe

To a solution of 3-phenylpropionyl-Pro-Lys(Cbz)Ψ[CHOHCO]—OMe (240 mg) inmethanol (5 ml) was added 10% palladium on charcoal (50 mg) and 216 ml2N hydrogen chloride solution. The mixture was hydrogenated atatmospheric pressure and at room temperature for 1 h. The palladiumcatalyst was removed by filtration and the solvent removed byevaporation at reduced pressure yielding3-phenylpropionyl-Pro-LysΨ[CHOHCO]—OMe quantitatively. TLC: R_(f)=0.13,silica gel, dichloromethane/methanol=9/1 v/v.

3-Phenylpropionyl-Pro-Lys(Boc)Ψ[CHOHCO]—OMe

To a solution of phenylpropionyl-Pro-LysΨ[CHOHCO]—OMe (196 mg.) in dryDMF (5 ml) was added bis(tert-butyl)anhydride (102 mg) and the pH wasadjusted to pH 8.5 by adding triethylamine. The reaction mixture wasstirred at room temperature overnight. The mixture was evaporated invacuo and the resulting residue was purified by chromatography on silica(eluent: dichloromethane/methanol=98/2 v/v to yield3-phenylpropionyl-Pro-Lys(Boc)Ψ[CHOHCO]—OMe (189 mg). TLC: R_(f)=0.43,silica gel, dichloromethane/methanol=9/1 v/v.

3-Phenylpropionyl-Pro-Lys(Boc)Ψ[CHOHCO]—OH

Phenylpropionyl-Pro-Lys(Boc)Ψ[CHOHCO]—OMe (185 mg.) was dissolved indioxane/water=7/3 (5 ml) and treated with 2M sodium hydroxide solution(267 ml) portionwise over 30 min at room temperature, keeping the pH at10-10.5. After 30 min the reaction mixture was diluted with water (20ml), 2M hydrogen chloride solution was added until pH 2.0 and the waterlayer was extracted with dichloromethane. The combined organic phaseswere washed with water (50 ml), brine (50 ml) and dried over sodiumsulfate, filtered and concentrated in vacuo to yield3-phenylpropionyl-Pro-Lys(Boc)Ψ[CHOHCO]—OH (228 mg). TLC: R_(f)=0.60,silica gel, EPAW=63/20/6/11 v/v/v/v.

3-Phenylpropionyl-Pro-Lys(Boc)Ψ[COCO]—OH

To a solution of phenylpropionyl-Pro-Lys(Boc)Ψ[CHOHCO]—OH (220 mg) indry dichloromethane (5 ml) were added 255 mg of periodinane (Dess-Martinreagent). After 1 h stirring at room temperature, 2% sodium thiosulfatesolution was added (15 ml) and the mixture was stirred for 30 min atroom temperature. The organic layer was separated, washed with water,dried over sodium sulfate, filtered and evaporated in vacuo to givecrude keto-acid 3-phenylpropionyl-Pro-Lys(Boc)Ψ[COCO]—OH (411 mg). TLC:R_(f)=0.47, silica gel, EPAW=63/20/6/11 v/v/v/v.

3-Phenylpropionyl-Pro-LysΨ[COCO]—OH

3-Phenylpropionyl-Pro-Lys(Boc)Ψ[COCO]—OH (411 mg) was treated with 90%trifluoroacetic acid/water for 1 h at room temperature. The reactionmixture was concentrated in vacuo and the residue was dissolved in waterand directly chraged onto a preparative HPLC Supelcosil LC-18-DB columnusing a gradient elution system of 20% A/70% B/10% C to 20% A/0% B/80% Cover 45 min, at a flow rate of 20 ml/min. (A: 0.5M phosphate buffer pH2.1, B: water, C: acetonitril/water=3/2). Yield: 71 mg. of3-phenylpropionyl-Pro-LysΨ[COCO]—OH. R_(t) (LC): 24.9 min; 20%A/80%B to20%A/20%B/60%C in 40 min.

In a similar manner as described above were prepared:

(a). 3,3diphenylpropionyl-Pro-LysΨ[COCO]—OH

R_(t) (LC): 36.42 min; 20% A/80% B/0% C to 20% A/20% B/60% C in 40 min.

(b). 3-Phenylpropionyl-(N-cyclopentyl)-Gly-LysΨ[COCO]—OH

R_(t) (LC): 34.29 min; 20% A/80% B/0% C to 20% A/20% B/60% C in 40 min.

(c). 3-[(p—Cl)-phenyl]propionyl-(N-cyclopentyl)-Gly-LysΨ[COCO]—OH

R_(t) (LC): 39.52 min; 20% A/80% B/0% C to 20% A/20% B/60% C in 40 min.

(d). 3-[(p—Cl)-phenyl]propionyl-Pro-LysΨ[COCO]—OH

R_(t) (LC): 31.31 min; 20% A/80% B/0% C to 20% A/20% B/60% C in 40 min.

(e). Naphthylsulfonyl-Asp-Pro-LysΨ[COCO]—OH

R_(t) (LC): 30.45 min; 20% A/80% B/0% C to 20% A/20% B/60% C in 40 min.

EXAMPLE 5 H—D-Cha-Pro-Lys-(2-benzothiazolyl)Boc-Lys(Cbz)-Ψ[CHOH]-(2-benzothiazolyl)

To a cold (−78° C.) solution of Boc-Lys(Cbz)-OMe (1 g) indichloromethane (25 ml) was added dropwise diisobutyl aluminumhydride(DiBAL-H; 7.6 ml of a 1M solution in hexane) whereby the reactiontemperature was kept below −70° C. The resulting solution was stirred at−78° C. for 30 min. A 5% citric-acid solution was added to the reactionmixture. The two layers mixture was stirred at room temperature for 10min, the layers were separated and the aqueous layer is was extractedtwice with dichloromethane. The combined dichloromethane layers werewashed with water, 5% sodium hydrogencarbonate, water and dried oversodium sulfate and filtered. The solution was placed under nitrogen and2-(trimethylsilyl)benzothiazole (0.79 g) was added and the reactionmixture was stirred for 16 h at room temperature. After evaporation todryness the residue was dissolved in dry tetrahydrofuran (15 ml) andtetrabutyl ammoniumfluoride (3.8 ml of a 1M solution in THF) was added.The mixture was stirred for 2 h at room temperature and water was added.The product was extracted with dichloromethane and purified bychromatography on silica (eluent: dichloromethane/ethyl acetate; 9/1v/v) to yield 724 mg of Boc-Lys(Cbz)-Ψ[CHOH]-(2-benzothiazolyl). TLC:R_(f)=0.35, silica gel, dichloromethane/ethyl acetate=7/3 v/v.

Boc-Lys(Cbz)-(2-benzothiazolyl)

To a solution of Boc-Lys(Cbz)-Ψ[CHOH]—(2-benzothiazolyl) (700 mg) in drydichloromethane (10 ml) was added 1 g. of periodinane (Dess-Martinreagent). After stirring for 1 h at room temperature, 2% sodiumthiosulfate solution was added and the mixture was stirred for another30 min at room temperature. The organic layer was separated, washed withwater, dried over sodium sulfate, filtered and evaporated in vacuo. Theresidue was purified by chromatography on silica (eluent: ethylacetate/heptane; 3/1 v/v) to yield 193 mg ofBoc-Lys(Cbz)-(2-benzothiazolyl). TLC: R_(f)=0.85, silica gel, ethylacetate/heptane=3/1 v/v.

Boc-D-Cha-Pro-Lys(Cbz)-(2-benzothiazolyl)

Boc-Lys(Cbz)-(2-benzothiazolyl) (193 mg) was dissolved in 50%TFA/dichloromethane (2 ml) and stirred for 1 h. at room temperature. Thecrude amine was isolated in quantitative yield after removal of thesolvent by evaporation, and used immediately to prepareBoc-D-Cha-Pro-Lys(Cbz)-(2-benzothiazolyl). Boc-D-Cha-Pro-OH wasdissolved in dry dimethylformamide (4 ml). After addition ofdiisopropylethylamine (DIPEA, 66 ml), the reaction mixture was placedunder nitrogen and cooled to −15° C. Isobutylchloroformate (50 ml) wassubsequently added and the mixture was allowed to stir for 15 min at−15° C. The crude amine was dissolved in dry DMF (4 ml), neutralisedusing diisopropylethylamine and added dropwise to the cold mixedanhydride solution. The reaction was stirred for 1 h at −15° C. and thenkept at 0° C. overnight. The mixture was evaporated to dryness. Theresidue was dissolved in ethyl acetate and successively washed withwater, 5% sodium hydrogencarbonate, water and brine, dried over sodiumsulfate and concentrated in vacuo. The residue was purified bychromatography on silica (eluent: ethyl acetate/heptane; 1/1 v/v) toyield Boc-D-Cha-Pro-Lys(Cbz)-(2-benzothiazolyl) (191 mg). TLC:R_(f)=0.66, silica gel, ethyl acetate/heptane=3/1 v/v.

H—D-Cha-Pro-Lys-(2-benzothiazolyl)

Boc-D-Cha-Pro-Lys(Cbz)(2-benzothiazolyl) was treated withtrifluoroacetic acid/thioanisole 10/1 v/v (2.2 ml) for 3.5 h at roomtemperature. The reaction mixture was concentrated in vacuo and theresidue was dissolved in water. The aqueous phase was washed extensivelywith diethyl ether. The water layer, containingH—D-Cha-Pro-Lys-(2-benzothiazolyl) was charged directly onto apreparative HPLC Supelcosil LC-18-DB column using a gradient elutionsystem of 20% A/80% B to 20% A/20% B/60% C over 40 min, at a flow rateof 20 ml/min. (A: 0.5 mM sodium phosphate buffer pH 2.1, B: water, C:acetonitrile/water; 3/2 v/v). Yield: 98 mg ofH—D-Cha-Pro-Lys-(2-benzothiazolyl). R_(t) (LC): 42 min 20% A/80% B to20% A/20% B/60% C in 40 min.

EXAMPLE 6 H—D-Cha-Pro-Lys-(2-tetrazolyl)Boc-Lys(Cbz)Ψ(CHOAc)-(2-tetrazolyl)

To a solution of Boc-Lys(Cbz)Ψ[cyanoacetate] (5.4 g) in 39 mldimethylformamide was added 801 mg of ammonium chloride and 975 mg ofsodium azide. The reaction mixture was heated to 80° C. and stiffedunder nitrogen during 48 h. Precipitated salt was filtered off and thesolution was evaporated under reduced pressure to dryness. This yielded4.9 g of the desired compound. TLC: Rf=0.42, silica gel,toluene/ethanol=6/4 v/v.

Boc Lys(Cbz)Ψ(CHOH)-(2-tetrazolyl)

Boc-Lys(Cbz)Ψ(CHOAc)-(2-tetrazolyl) (1.25 g) was dissolved in 60 ml ofdioxane/water 7/3 and 2.65 ml of a 2N sodium hydroxide solution wasadded. The solution was stirred at room temperature during 2.5 h afterwhich the reaction appeared completed. The pH was adjusted to 5 and theresulting mixture was evaporated to dryness under reduced pressure. Theresidue was dissolved in methanol/dichloromethane 1/1 and insoluble saltwas filtered off. This resulted in 1.27 g of the deacetylated product.TLC: Rf=0.40, silica gel, toluene/ethanol=6/4 v/v.

Boc-Lys(Cbz)-(2-tetrazolyl)

To a solution of 0.56 g of Boc-Lys(Cbz)Ψ(CHOH)-(2-tetrazolyl) in 37 mldry dichloromethane, 1.38 g of Dess-Martin periodane reagent was added.The mixture was stirred at room temperature for 30 minutes after whichthe reaction was quenched with a 10% sodium thiosulfate solution inwater. The organic layer was extracted with water and sodiumhydrogencarbonate (5% in water). the water layers were combined enextracted with 1-butanol. The 1-butanol layer was evaporated to drynessunder reduced pressure. The residue was chromatographed on a silica gelcolumn using the eluent: ethyl acetate/pyridine/aceticacid/water=263/20/6/11 v/v/v/v. Yield: 0.22 g. TLC: Rf=0.30, silica gel,ethyl acetate/pyridine/acetic acid/water=63/20/6/11 v/v/v/v.

H-Lys(Cbz)-(2-tetrazolyl).TFA

Boc-Lys(Cbz)-(2tetrazolyl) (0.21 g) was dissolved in 16 mltrifluoroacetic acid/water 9/1. The mixture was stirred for 1 h at roomtemperature after which the solution was concentrated in vacuo to yieldan oil. Yield: 0.34 g, used immediately to prepare tripeptideBoc-D-Cha-Pro-Lys(Cbz)-(2-tetrazolyl).

Boc-D-Cha-Pro-Lys(Cbz)-(2-tetrazolyl)

Boc-D-Cha-Pro-OH (0.19 g) was prepared according to the proceduredescribed in example 1 for the dipeptide moiety. The coupling toH-Lys(Cbz)-(2-tetrazolyl) (0.17 g) was performed in a similar manner asdescribed in example 5. Purification on silica gel (eluent: ethylacetate/pyridine/acetic acid/water=163/20/6/11 v/v/v/v) yielded 0.21 gof the desired compound. TLC: Rf=0.17, silica gel, ethylacetate/pyridine/acetic acid/water=63/20/6/11 v/v/v/v.

H—D-Cha-Pro-Lys-(2-tetrazolyl)

The removal of the protective groups and the HPLC purification wasperformed in an analogous procedure as described for example 5. Yield:20 mg.

Rt (LC): 23.3 and 24.5 min, 20% A, 80% B to 20% A, 20% B and 60% C in 40min.

EXAMPLE 7 HOOC—CH₂—CO—D-Cha-Pro-Lys-(2-thiazolyl)H—D-Cha-Pro-Lys(Cbz)(2-thiazolyl).TFA

Boc-D-Cha-Pro-Lys(Cbz)-(2-thiazolyl) was prepared according to theprocedure described for example 5. 0.30 g of this tripeptide wasdissolved in 3 ml of trifluoroacetic acid/dichloromethane 1/1 v/v andthe solution was stirred for 1 h at room temperature. The solution wasevaporated to dryness under reduced pressure and coevaporated threetimes with toluene. Yield: quantitative, oil, used immediately for thenext step. TLC: Rf=0.30, silica gel, ethyl acetate/pyridinelaceticacid/water=163/20/6/11 v/v/v/v.

(t-butyl-OOC—CH₂—CO)—D-Cha-Pro-Lys(Cbz)-(2-thiazolyl)

H—D-Cha-Pro-Lys(Cbz)2-thiazolyl).TFA (0.33 g) was dissolved in 3 ml ofdry dichloromethane and 76 mg of mono-tertiary butyl malonate was addedand the pH was adjusted to approximately 8 with triethylamine. Next,benzotriazolyloxytris(dimethylamino)-phosphonium hexafluorophosphate(211 mg) was added and the reaction mixture was stirred at roomtemperature for 2 h and at 4° C. for the next 16 h. The solution wasconcentrated in vacuo, dissolved in ethyl acetate and washed three timeswith water and brine. The organic layer was again concentrated in vacuoafter being dried on magnesium sulfate. The residue was chromatographedon silica using heptane/ethyl acetate 2/8 v/v as eluent. This yielded270 mg of the acylated tripeptide. TLC: Rf=0.21, silica gel,heptane/ethyl acetate=8/2 v/v.

HOOC—CH₂—CO—D-Cha-Pro-Lys-(2-thiazolyl)

The removal of the protective groups and the HPLC purification wereperformed in an analogous procedure as described for example 5. Yield:124 mg.

Rt (LC): 38.23 min, 20% A, 80% B to 20% A, 20% B and 60% C in 40 min.

EXAMPLE 8 HOOC—(CH₂)₂CO—D-Cha-Pro-Lys-(2-thiazolyl)(HOOC—(CH₂)₂—CO)—D-Cha-Pro-Lys(Cbz)-(2-thiazolyl)

H—D-Cha-Pro-Lys(Cbz)-(2-thiazolyl).TFA (0.33 g) was dissolved in 3 ml ofdry dichloromethane and 0.335 ml of pyridine. To this solution 48 mg ofsuccinic anhydride was added and the resulting solution was stirred atroom temperature under nitrogen. After 4 h the reaction appearedcompleted and was quenched with a few droplets of water. The mixture wasconcentrated in vacuo, dissolved in ethyl acetate, washed with dilutedacid, water and brine and dried on magnesium sulfate. After removal ofthe salt the organic layer was concentrated in vacuo which resulted in320 mg of an oil.

TLC: Rf=0.37, silica gel, dichloromethane/methanol 9/1 v/v.

HOOC—(CH₂)₂—CO—D-Cha-Pro-Lys-(2-thiazolyl)

The removal of the protective groups and the HPLC purification wereperformed in an analogous procedure as described for example 5. Yield:187 mg.

Rt (LC): 38.31 min 20% A, 80% B to 20% A, 20% B and 60% C in 40 min.

EXAMPLE 9 HOOC—CH(CH₃)—D-Cha-Pro-Lys-(2-thiazolyl)(t-Butyl-OOC—CH(CH₃))—D-Cha-Pro-Lys(Cbz)-(2-thiazolyl)

H—D-Cha-Pro-Lys(Cbz)-(2-thiazolyl).TFA (0.33 g) was dissolved in 2 ml ofacetonitril. Next, 0.50 g of 2-bromopropionic acid tert-butylester wasadded followed by 25 mg of sodium iodide. The pH of the solution wasadjusted to 8 with diisopropylethylamine and kept at that basicity for12 days at room temperature. The reaction mixture was concentrated invacuo, dissolved in ethyl acetate, washed with water, dried on magnesiumsulfate and again concentrated. The residue was chromatographed onsilica using ethyl acetate/toluene 1/1 v/v as eluent. Yield: 279 mg.TLC: 0.75, silica gel, ethyl acetate.

HOOC—CH(H₃)—D-Cha-Pro-Lys-(2-thiazolyl)

The removal of the protective groups and the HPLC purification wereperformed in an analogous procedure as described for example 5. Yield:40 mg and 29 mg (separated diastereomers). Rt (LC): 30.06 min and 34.87min (separated diastereomers), 20% A, 80% B to 20% A, 20% B and 60% C in40 min.

EXAMPLE 10 HOOC—(CH₂)₂—D-Cha-Pro-Lys-(2-thiazolyl)(t-Butyl-OOC—(CH₂)₂—D-Cha-Lys(Cbz)-(2-thiazolyl)

H—D-Cha-Pro-Lys(Cbz)-(2-thiazolyl).TFA (0.21 g) was dissolved in 5 ml ofacetonitril. Next, 1.84 ml of acrylacid tert-butylester was added inthree portions. The pH of the solution was adjusted to 8 withdiisopropylethylamine and kept at that basicity for 13 days at roomtemperature. The reaction mixture was concentrated in vacuo, dissolvedin ethyl acetate, washed with water, dried on magnesium sulfate andagain concentrated. The residue was chromatographed on silica usingethyl acetate/toluene 2/1 v/v as eluent. Yield: 92 mg. TLC: Rf=0.62,silica gel, ethyl acetate.

HOOC—(CH₂)₂—D-Cha-Pro-Lys-(2-thiazolyl)

The removal of the protective groups and the HPLC purification wereperformed in an analogous procedure as described for example 5. Yield:40 mg.

Rt (LC): 32.83 min, 20% A, 80% B to 20% A, 20% B and 60% C in 40 min.

EXAMPLE 11 N—Me—D-norLeu-Pro-Lys-(2-thiazolyl) H—D-norLeu-OMe. HCl

To 270 ml of methanol, cooled to −15° C., 18.2 g of thionylchloride wasadded. Subsequently, the temperature was allowed to rise to −10° C. thankept constant for 20 min after which 10 g H—D-norLeu-OH was added. Thetemperature was slowly increased and at reflux kept constant for 5 h.The product was crystallized from methanol and diethyl ether at 4° C.and this yielded 12.9 g. TLC: Rf=0.61, silica gel, n-butanol/aceticacid/water 10/1/3 v/v/v.

Boc-D-norLeu-OMe

H—D-norLeu-OMe.HCl (12.9 g) was dissolved in 200 ml of dry methanolfollowed by addition of di-tert-butyl dicarbonate (15.5 g) andtriethylamine (19.8 ml). The reaction was stirred for 3h at roomtemperature after which the mixture was concentrated in vacuo. Next, theresidue was dissolved in ethyl acetate and washed with water. Theproduct was chromatographed on silica using heptane/ethyl acetate 3/1v/v . Yield: 16.9 g. TLC: Rf=0.55, silica gel, heptane/ethyl acetate 3/1v/v.

N—Me-Boc-D-norLeu-OMe

Boc-D-norLeu-OMe (16.9 g) was dissolved in 200 ml of drydimethylformamide under nitrogen. Next, methyliodide (24.9 ml) wasadded, cooled to 0° C., sodium hydride (3.31 g) was added and themixture was allowed to react during 16 h at room temperature. Themixture was concentrated in vacuo, dissolved in ethyl acetate, washedwith diluted hydrogen chloride (0.1 N), water, sodium hydrogencarbonate(5%) and water, dried and concentrated again. This yielded 18.8 g ofalkylated product. TLC:=0.56, silica gel, heptane/ethyl acetate 3/1 v/v.

N—Me-Boc-D-norLeu-OH

N—Me-Boc-D-norLeu-OMe (18 g) was dissolved in 400 ml of dioxane/water9/1 v/v and the pH of the solution was adjusted to 12 with 1N sodiumhydroxide. The reaction was allowed to proceed for 2 h, keeping the pHconstant at 12. The work-up procedure involved a pH adjustment to 2,cooling on ice, adding extra water (400 ml) and an extraction withdichloromethane. The organic layer was washed with brine, dried,filtered and concentrated in vacuo. This yielded 18.9 g of product whichcontained some dioxane.

TLC:=0.26, silica gel, dichloromethane/methanol 9/1 v/v.

N—Me-Boc-D-norLeu-Pro-OH.

First the N-succinimide ester was prepared starting fromN—Me-Boc-D-norLeu-OH. 18 g of this derivative was dissolved inacetonitrile (250 ml), and then EDCI (14.5 g) and N-hydroxy-succinimide(HONSu) (8.7 g) were added. The reaction required 16 h at roomtemperature after which the solvent was removed, the residue wasdissolved in ethyl acetate and washed with water and dried. This yielded24.3 g of crude ONSu ester. The next step was to dissolve proline.HCl(20.9 g) in 300 ml dimethylformamide and 300 ml water and the pH wasadjusted to 8 with 2N sodium hydroxide solution. A solution of the ONSuester (24.3 g in 300 ml of dimethylformamide) was added dropwise to thissolution keeping the pH constant. The reaction was completed after 5 h,after which the organic solvent was largely removed by evaporation underreduced pressure. Extra water (300 ml) was added and the pH was adjustedto 2. The product was extracted with ethyl acetate and washed withwater. After drying, filtration and concentration the product wasobtained as a yellow oil in 22.2 g. The crude product waschromatographed on silica using ethyl acetate/methanol 8/2 v/v aseluent. Yield: 13.2 g.

TLC: Rf=0.65, silica gel, ethyl acetate/pyridine/aceticacid/water=163/20/6/11 v/v/v/v.

N—Me—D-norLeu-Pro-Lys-(2-thiazolyl).

The mixed anhydride coupling between N—Me-Boc-D-norLeu-Pro-OH andH-Lys(Cbz)-(2-thiazolyl).TFA, the deprotection and the purification weredone according to the procedures described in example 5. Yield: 107 mg.

Rt (LC): 23.22 min, 20% A, 80% B to 20% A, 20% B and 60% C in 40 min.

EXAMPLE 12 N—Me—D-Cha-Pro-Lys-(2-thiazolyl)

All steps leading to this tripeptide were performed in a similar manneras described for example 11, and starting with Boc-D-Cha-OH. Yield: 253mg.

Rt (LC): 31.82 min, 20% A, 80% B to 20% A, 20% B and 60% C in 40 min.

EXAMPLE 13 N—Me—D-Phe-N-cyclopentyl-Gly-Lys-(2-thiazolyl)N—Me-Boc-D-Phe-N-cyclopentyl-Gly-OMe

N—Me-Boc-D-Phe-OH (prepared according to example 11) (26 g) andN-cyclopentyl-Gly-OMe (21 g, see example 3) were dissolved in 300 ml ofdimethylformamide. Next, TBTU (36 g) was added and the pH was adjustedto 8 with diisopropylethylamine (20 ml). The reaction mixture wasstirred for 16 h and than concentrated in vacuo, dissolved in ethylacetate, washed with sodium hydrogencarbonate (5%) and brine, dried onmagnesium sulfate and again concentrated in vacuo. Yield: 24.8 g. TLC:Rf=0.62, silica gel, dichloromethane/methanol 95/5 v/v.

N—Me-Boc-D-Phe-N-cyclopentyl-Gly-OH

N—Me-Boc-D-Phe-N-cyclopentyl-Gly-OMe (17.3 g) was dissolved in 150 ml oftetrahydrofuran/water 135/15 v/v and 4 g of sodium hydroxide (in water)was added. After 2 h the reaction was stopped by adjusting the pH to 2and the product was extracted with dichloromethane. After washing withwater, drying on magnesium sulfate, concentrating in vacuo andcrystallisation from dichloromethane/diethyl ether, the reaction yielded13.1 g.

TLC: Rf=0.52, silica gel, dichloromethane/methanol 9/1 v/v.

N—Me-D-Phe-N-cyclopentyl-Gly-Lys-(2-thiazolyl)

The next steps were done according to the procedure described forexample 11. Yield: 110 mg.

Rt (LC): 33.43 min, 20% A, 80% B to 20% A, 20% B and 60% C in 40 min.

EXAMPLE 14 N—Me-D-Phe-Pro-Lys-(2-thiazolyl)

N—Me-Boc-D-Phe-Pro-OH was prepared according to the description forexample 1. The mixed anhydride coupling to H-Lys(Cbz)-(2-thiazolyl), thedeprotection and the purification were done according to example 5.Yield: 148 mg.

Rt (LC): 27.22 min, 20% A, 80% B to 20% A, 20% B and 60% C in 40 min.

EXAMPLE 15 3,3-Diphenylpropionyl-Pro-Lys(ethoxycarbonyl)-(2-thiazolyl)

3,3-Diphenylpropionyl-Pro-Lys-(2-thiazolyl) was prepared as described inexample 1. A solution was made of 20 mg of this dipeptide indioxane/water 4/1 (4 ml) and the pH was adjusted with 1 N sodiumhydroxide to 12. Next, 22 mg of ethylchloroformiate was added and thesolution was stirred for 2 h at room temperature. The mixture wasdiluted with water and extracted with dichoromethane, washed with water,dried on magnesium sulfate, concentrated in vacuo and finally,freezedried from tert-butanol/water 1/1 v/v. Yield: 15 mg. TLC: Rf=0.92,silica gel, ethyl acetate/pyridine/acetic acid/water=63/20/6/11 v/v/v/v.

EXAMPLE 16 HOOC—CH₂—D-Cha-Pro-Lys-(2-oxazolyl)Boc-Lys(Cbz)Ψ[CHOH]-(2-oxazolyl)

To a solution of 0.975 g of Boc-Lys(Cbz)-OMe in 25 ml of dichloromethaneat −78° C. under a nitrogen atmosphere was added 6 ml of a 1Mdiisobutylaluminiumhydride solution in hexane. After 15 min the reactionwas completed, the reaction mixture poured into 150 ml of 2% citric acidsolution and filtered. The organic layer was separated, washed withwater and brine, dried (magnesium sulfate) and concentrated. The residuewas coevaporated with toluene to give 0.92 g of Boc-Lys(Cbz)-H. Thisaldehyde (0.89 g) was dissolved in 1.4 ml of toluene and 0.90 g of2-(trimethylsilyl)oxazole (prepared according to: Edwards, P. D.,Wolanin, D. J., Andisik D. W., and Davis W., J. Med. Chem. 38, 76(1995)) was added and heated at 80° C. After 60 h the reaction mixturewas concentrated, the residue dissolved in 5 ml of tetrahydrofuran,treated with 3 ml of a 3M tetrabutylammonium fluoride in tetrahydrofuransolution and stirred at room temperature for 2 h. The mixture wasconcentrated dissolved in ethyl acetate, washed with 3% aqueous sodiumhydrogencarbonate solution and brine, dried (magnesium sulfate) andevaporated. Purification by column chromatography on silica gel elutingwith a gradient of ethyl acetate/dichloromethane=2/1 (v/v) to ethylacetate afforded an oil that was rechromatographed on silica gel elutingwith a gradient of ethyl acetate/heptane=1/1 (v/v) to ethylacetate/heptane=1/3 (v/v) to give 0.22 g of the title compound.

TLC: Rf=0.7, silica gel, ethyl acetate.

Boc-Lys(Cbz)-(2-oxazolyl)

To a solution of 0.22 g of Boc-Lys(Cbz)-Ψ[CHOH]—(2-oxazolyl) in 10 ml ofdichloromethane was added 0.22 g of periodiane (Dess-Martin reagent).After 1.5 h stirring at room temperature 10 ml of aqueous 5% sodiumthiosulfate was added and the mixture was stirred for 15 min at roomtemperature. The organic layer was separated, washed with water, aqueous5% sodium hydrogen carbonate and brine, dried over magnesium sulfate andconcentrated. Purification by chromatography on silica gel eluting withheptane/ethyl acetate=1/1 (v/v) yielded 162 mg of the title compound.TLC: Rf=0.5, silica gel, heptane/ethyl acetate=1/3 (v/v).

(tBuOOCCH₂)(Boc)-D-Cha-Pro-Lys(Cbz)-(2-oxazolyl)

The procedure described for example 5 was used. Deprotection of 0.16 gof Boc-Lys(Cbz)-(2-oxazolyl) and coupling with 0.19 g of(tBuOOCCH₂)(Boc)-D-Cha-Pro-OH afforded 0.19 g of(tBuOOCCH₂)(Boc)-D-Cha-Pro-Lys(Cbz)-(2-oxazolyl).

TLC: Rf=0.3, silica gel, heptane/ethyl acetate=1/3 (v/v).

HOOCCH₂—D-Cha-Pro-Lys-(2-oxazolyl)

The procedure described for example 5 was used. 0.19 g of(tBuOOCCH₂)(Boc)-D-Cha-Pro-Lys(Cbz)-(2-oxazolyl) afforded 52 mg of thetitle compound.

Rt (LC): 28.46 min, 20% A/80% B to 20% A/20% B/60% C in 40 min.

EXAMPLE 17 EthylSO₂-norLeu(cyclo)Gly-Lys-(2-thiazolyl)

Boc-L-α-amino-ε-caprolactam

To a stirred solution of L-α-amino-ε-caprolactam (10 g) in dioxane/water(2/1 v/v) (30 ml) was added 1N sodium hydroxide solution (7.8 ml)followed by di-t-butyl carbonate (18.8 g). The mixture was stirred for16 h. at room temperature and concentrated in vacuo. The residue wasdissolved in ethyl acetate and washed with water and brine, dried oversodium sulfate, filtered and evaporated in vacuo. The crude material wastriturated by hexane, filtered and dried in vacuo to yieldBoc-L-α-amino-ε-caprolactam (16 g).

TLC: Rf=0.85, ethyl acetate/heptane 1/1 v/v on silica.

Boc-norLeu(cyclo)Gly-OMe

Boc-L-α-amino-ε-caprolactam (10 g) was dissolved in dichloromethane (100ml). At −20° C. a 1M solution of bis(trimethylsilyl)amide inTHF/cyclohexane (1/1 v/v) (1 equiv.) was added slowly and the mixturewas stirred for 30 min. Methyl bromoacetate (4 ml) was subsequentlyadded and the mixture was stirred for 2 h. at room temperature.Additional bis(trimethylsilyl)amide in THF/cyclohexane (1/1 v/v) wasadded to force the reaction to completion. The mixture was diluted bydichloromethane and washed with 0.1 N hydrochloric acid solution, water,5% aqueous sodium hydrogencarbonate solution and brine, dried oversodium sulfate, filtered and evaporated in vacuo. The residue waspurified by chromatography on silica (eluent: heptane/ethyl acetate 6/4v/v to yield Boc-norLeu(cyclo)Gly-OMe (12 g).

TLC: Rf=0.55, ethyl acetate/heptane 6/4 v/v on silica.

EthylSO₂-norLeu(cyclo)Gly-OMe

Boc-norLeu(cyclo)Gly-OMe (3 g) was dissolved in 50% TFA/dichloromethane(30 ml) and stirred for 1 h. at room temperature. The reaction mixturewas evaporated in vacuo. The crude amine was dissolved indichloromethane (30 ml) and a solution of ethanesulphonylchloride (1.29g) in dichloromethane (10 ml) was added slowly at 0° C. Triethylaminewas added to keep the pH at 8 during the reaction. The mixture wasstirred for 1 h at room temperature, whereafter the mixture wasconcentrated in vacuo. The residue was dissolved in ethyl acetate andwashed with 5% sodium hydrogencarbonate solution, water and brine, driedover sodium sulfate, filtered and evaporated in vacuo. The residue waspurified by chromatography on silica (eluent: dichloromethane/ethylacetate 95/5 v/v%. to yield ethylSO₂-norLeu(cyclo)Gly-OMe (1.45 g). TLC:Rf=0.30, dichloromethane/ethyl acetate 9/1 v/v on silica.

EthylSO₂-norLeu(cyclo)Gly-OH

A solution of ethylSO₂-norLeu(cyclo)Gly-OMe (1.45 g) in 50 ml ofdioxane/water 9/1 v/v was treated with sufficient 1N sodium hydroxide tokeep the pH at 13 for 2 hours at room temperature. After acidification,the mixture was poured into water and extracted with dichloromethane.The organic layer was washed with water and dried on sodium sulfate. Thefiltrate was evaporated and yielded 600 mg of the title compound.

TLC Rf=0.45, ethyl acetate/pyridine/acetic acid/water 63/20/6/11 v/v/v/von silica.

EthylSO₂-norLeu(cyclo)Gly-Lys(Cbz)-(2-thiazolyl)

EthylSO₂-norLeu(cyclo)Gly-OH (482 mg) was dissolved in drydimethylformamide (5 ml). After addition of ethyl diisopropyl amine(0.36 ml), the reaction mixture was placed under nitrogen and cooled to−20° C. Isobutylchloroformate (140 ml) was subsequently added and themixture was allowed to stir for 15 min at −20° C.H-Lys(Cbz)-(2-thiazolyl).TFA was dissolved in dry dimethylformamide (3ml) and added dropwise to the cold mixed anhydride solution, maintainingthe pH at 8.5 by addition of ethyl diisopropyl amine. The reactionmixture was stirred for 15 min at −20° C. The reaction mixture wasevaporated to dryness. The residue was dissolved in ethyl acetate andsuccessively washed with 5% aqueous sodium hydrogencarbonate solution,water and brine, dried over sodium sulfate and concentrated in vacuo.The residue was purified by chromatography on silica (eluent:dichloromethane/methanol=95/5 v/v) to yieldEthylSO₂-norLeu(cyclo)Gly-Lys(Cbz)-(2-thiazolyl)(607 mg).

TLC: Rf=0.63, ethyl acetate/pyridine/acetic acid/water 60/3/1/2 v/v/v/von silica.

EthylSO₂-norLeu(cyclo)Gly-Lys-(2-thiazolyl)

EthylSO₂-norLeu(cyclo)Gly-Lys(Cbz)-(2-thiazolyl) (600 mg) was treatedwith trifluoroacetic acid/thioanisole 10/1 v/v (10 ml) for 4 h at roomtemperature. The reaction mixture was concentrated in vacuo and theresidue was dissolved in water. The aqueous phase was washed extensivelywith diethyl ether. The water layer was concentrated in vacuo,coevaporated with diluted hydrochloric acid and lyophilised from water.The crude product was charged onto a preparative HPLC Deltapack C18 RPcolumn using a gradient elution system of 200/4 A/80%B to 20% A/40%B/40% C over 40 minutes, at a flow rate of 50 ml/min. Yield: 233 mg ofethylSO₂-norLeu(cyclo)Gly-Lys-(2-thiazolyl).

R_(t) (LC): 26.73 min. 20% A/80%B to 20% A/20% B/60% C in 40 minutes.

EXAMPLE 18 BenzylSO₂-norLeu(cyclo)Gly-Lys-(2-thiazolyl)

This compound was prepared in a similar manner as described in Example17.

R_(t) (LC): 37.05 min. 20% A/80%B to 20% A/20% B/60% C in 40 minutes.

EXAMPLE 197-Methoxy-2-Napthylsulphonyl-norLeu(cyclo)Gly-Lys-(2-thiazolyl)

This compound was prepared in a similar manner as described in Example17.

R_(t) (LC): 26.40 min. 20% A/60%B/20% C to 100% C in 40 minutes.

EXAMPLE 20(4aR,8aR)-perhydroisoquinoline-1(R)-carbonyl-Pro-Lys-(2-thiazolyl)2-Cbz-(4a,8aR)-perhydroisoquinoline-1(R,S)-carboxylic acid

2-Cbz-(4aR,8aR)-perhydroisoquinoline-1(R,S)-carboxylic acid has beensynthesized as described in EP0643073, example 1.

TLC: Rf=0.85, ethyl acetate/pyridine/acetic acid/water 63/20/6/11v/v/v/v on silica.

2-Cbz-(4aR,8aR)-perhydroisoquinoline-1(R,S)-carbonyl-Pr-O-tBu

To a cold solution (0° C.) of2-Cbz-(4aR,8aR)-perhydroisoquinoline-1(R,S)-carboxylic acid (500 mg) indimethylformamide (5 ml) were successively added DCCI(1,3-dicyclohexylcarbodiimide; 342 mg), HOBT (1-hydroxybenzotriazolehydrate; 319 mg), H-Pro-OtBu (270 mg) and triethylamine (0.55 ml). Thereaction mixture was stirred at 0° C. for 1 h. and then kept at roomtemperature overnight. The reaction mixture was cooled to −20° C. andthe DCU (1,3-dicyclohexylurea) was removed by filtration. The filtratewas concentrated in vacuo and the residue was dissolved in ethylacetate. This solution was washed successively with 5% aqueous sodiumhydrogencarbonate solution, 3% aqueous citric acid solution, water andbrine, dried over sodium sulfate and concentrated in vacuo. The residuewas purified by chromatography on silica (eluent: heptane/ethyl acetate4/1 v/v) to yield2-Cbz-(4aR,8aR)-perhydroisoquinoline-1(R,S)-carbonyl-Pro-O-tBu)(634 mg).

TLC: Rf=0.90, ethyl acetate/pyridinelacetic acid/water 63/20/6/11v/v/v/v on silica.

2-Cbz-(4aR,8aR)-perhydroisoquinoline-1(R,S)-carbonyl-Pro-OH

2-Cbz-(4aR,8aR)-perhydroisoquinoline-1(R,S)carbonyl-Pro-O-t-butyl ester(600 mg) was stirred in a mixture of dichloromethane (1 ml),trifluoroacetic acid (3 ml), anisole (0.15 ml) for 1 h at roomtemperature. The reaction mixture was concentrated in vacuo at lowtemperature and the residue was dissolved in water at pH 9.5. Theaqueous phase was washed with diethyl ether, whereafter the aqueouslayer was acidified to pH 2.5 by 2M hydrochloric acid solution. Theaqueous layer was extracted with ethyl acetate and the organic phase waswashed with brine, dried over sodium sulfate and concentrated in vacuoto yield 2-Cbz-(4aR,8aR)-perhydroisoquinoline-1(R,S)-carbonyl-Pro-OH(588 mg). TLC: Rf=0.54, ethyl acetate/pyridine/acetic acid/water60/3/1/2 v/v/v/v on silica.

2-Cbz-(4aR,8aR)-perhydroisoquinoline-1(R,S)-carbonyl-Pro-Lys(Cbz)-(2-thiazolyl)

2-Cbz-(4aR,8aR)-perhydroisoquinoline-1(R,S)-carbonyl-Pro-OH (500 mg) wasdissolved in dry dimethylformamide (5 ml). After addition of ethyldiisopropyl amine (0.41 ml), the reaction mixture was placed undernitrogen and cooled to −20° C. Isobutylchloroformate (156 ml) wassubsequently added and the mixture was allowed to stir for 15 min at−20° C. H-Lys(Cbz)-(2-thiazolyl).TFA (594 mg) was dissolved in drydimethylformamide (3 ml) and added dropwise to the cold mixed anhydridesolution, maintaining the pH 8.5 by addition of ethyl diisopropyl amine.The reaction mixture was stirred for 15 min at −20° C. The reactionmixture was evaporated to dryness. The residue was dissolved in ethylacetate and successively washed with 5% aqueous sodium hydrogencarbonatesolution, water and brine, dried over sodium sulfate and concentrated invacuo. The residue was purified by chromatography on silica (eluent:dichloromethane/methanol=95/5 v/v %) to yield2-Cbz-(4aR,8aR)-perhydroisoquinoline-1(R,S)-carbonyl-Pro-Lys(Cbz)-(2-thiazolyl)(880mg). TLC: Rf=0.42, ethyl acetate/heptane 3/1 v/v on silica.

(4aR,8aR)-perhydroisoquinoline-1(R,S)-carbonyl-Pro-Lys-(2-thiazolyl)

(4aR,8aR)-perhydroisoquinoline-1(R,S)-carbonyl-Pro-Lys-(2-thiazolyl)(875 mg) was treated with trifluoroacetic acid/thioanisole 10/1 v/v (10ml) for 4 h at room temperature. The reaction mixture was concentratedin vacuo and the residue was dissolved in water. The aqueous phase waswashed extensively with diethyl ether. The water layer was concentratedin vacuo, coevaporated with diluted hydrochloric acid and lyophilisedfrom water. The crude product was charged onto a preparative HPLCDeltapack C18 RP column using a gradient elution system of 20% A/80%B to20% A/53% B/27% C over 40 minutes, at a flow rate of 50 ml/min. Yield:211 mg of(4aR,8aR)-perhydroisoquinoline-1(R)-carbonyl-Pro-Lys-(2-thiazolyl).

R_(t) (LC): 28 min. 20% A/80%B to 20% A/20% B/60% C in 40 minutes.

EXAMPLE 21 EthylSO₂—D-Cha-Pro-Lys-(2-thiazolyl) Boc-D-Cha-Pro-OBzl(Bzl=benzyl)

Boc-D-Cha-Pro-OBzl was prepared according a similar manner as describedin example 1 using Boc-D-Cha and Pro-OBzl.

TLC: Rf=0.5, dichloromethane/methanol 95/5 v/v on silica.

EthylSO₂—D-Cha-Pro-OBzl

Boc-D-Cha-Pro-OBzl (3.8 g) was dissolved in 50% TFA/dichloromethane (25ml) and stirred for 30 minutes at room temperature. The reaction mixturewas evaporated in vacuo. The crude amine was dissolved indichloromethane (50 ml) and ethanesulphonylchloride (0.8 ml) was addedat −78° C. Triethylamine was added to keep the pH 8 during the reaction.The mixture was stirred for 3 h at 0° C., whereafter water (25 ml) wasadded. After an additional stirring for 30 minutes at room temperature,the reaction mixture was concentrated in vacuo. The residue wasdissolved in diethyl ether and washed with 1N hydrochloric acidsolution, water, 5% sodium hydrogencarbonate solution and brine, driedover sodium sulfate, filtered and evaporated in vacuo. Trituration ofthe crude material with methanol yielded ethylSO₂—D-Cha-Pro-OBzl (3.0g). TLC: Rf=0.6, dichloromethane/methanol 95/5 v/v on silica.

EthylSO₂—D-Cha-Pro-OH

To a solution of EthylSO₂—D-Cha-Pro-OBzl (10 g) in tetrahydrofuran (250ml) was added 1M solution of tetrabutylammonium fluoride intetrahydrofuran (84 ml). The reaction mixture was stirred for 30 minutesat room temperature and poured into water (11). The aqueous solution wasextracted with ethyl acetate. The combined organic layers weresuccessively washed with 1N hydrochloric acid solution and water, driedover sodium sulfate and concentrated in vacuo. The residue was purifiedby crystallisation from ethyl acetate/diisopropylether to yieldEthylSO₂—D-Cha-Pro-OH (6.0 g).

TLC: Rf=0.2, ethyl acetate/pyridinelacetic acid/water 163/20/6/11v/v/v/v on silica.

EthylSO₂—D-Cha-Pro-Lys(Cbz)(2-thiazolyl)

EthylSO₂—Dha-Pro-OH (397 mg) was dissolved in dry dimethylformamide (3ml). After addition of ethyl diisopropyl amine (0.19 ml), the reactionmixture was placed under nitrogen and cooled to −20° C.Isobutylchloroformate (130 ml) was subsequently added and the mixturewas allowed to stir for 15 min at −20° C. H-Lys(Cbz)42-thiazolyl).TFAwas dissolved in dry dimethylformamide (3 ml) and added dropwise to thecold mixed anhydride solution, maintaining the pH at 8.5 by addition ofethyl diisopropyl amine. The reaction mixture was stirred for 15 min at−20° C. and 1 h at room temperature. The reaction mixture was evaporatedto dryness. The residue was dissolved in ethyl acetate and successivelywashed with 5% aqueous sodium hydrogencarbonate solution, water andbrine, dried over sodium sulfate and concentrated in vacuo. The residuewas purified by chromatography on silica (eluent: ethyl acetate/heptane2/1 v/v) to yield EthylSO₂—D-Cha-Pro-Lys(Cbz)-(2-thiazolyl)(575 mg).

TLC: Rf=0.32, ethyl acetate/heptane 2/1 v/v on silica.

EthylSO₂—D-Cha-Pro-Lys-(2-thiazolyl)

EthylSO₂—D-Cha-Pro-Lys(Cbz)-(2-thiazolyl) (570 mg) was treated withtrifluoroacetic acid/thioanisole 10/1 v/v (44 ml) for 4 h at roomtemperature. The reaction mixture was concentrated in vacuo and theresidue was dissolved in water. The aqueous phase was washed extensivelywith diethyl ether. The water layer was concentrated in vacuo,coevaporated with diluted hydrochloric acid and lyophilised from water.The crude product was charged onto a preparative HPLC Deltapack C18 RPcolumn using a gradient elution system of 20% A/80%B to 20% A/30% B/50%C over 40 minutes, at a flow rate of 80 mI/min. Yield: 275 mg ofEthylSO₂—D-Cha-Pro-Lys-(2-thiazolyl).

R_(t) (LC): 26.06 min. 20% A/60%B/20% C to 100% C in 40 minutes.

EXAMPLE 22 EthylSO₂—D-Phe-Pro-Lys-(2-thiazolyl) Boc-D-Phe-Pro-OBzl

This compound was prepared according a similar manner as described inexample 1 using Boc-D-Phe and Pro-OBzl.

TLC: Rf=0.9, ethyl acetate/pyridine/acetic acid/water 60/3/1/2 v/v/v/von silica

EthylSO₂—D-Phe-Pro-OH

This compound was prepared according a similar manner as described inexample 21 using Boc-D-Phe-Pro-OBzl.

TLC: Rf=0.48, ethyl acetate/pyridine/acetic acid/water 163/20/6/11v/v/v/v on silica.

EthylSO₂—D-Phe-Pro-Lys(Cbz)-(2-thiazolyl)

EthylSO₂—D-Phe-Pro-Lys(Cbz)2-thiazolyl) was prepared according a similarmanner as described in example 21 using EthylSO₂—D-Phe-Pro-OH andLys(Cbz)2-thiazolyl).

TLC: Rf=0.32, ethyl acetate/heptane 8/2 v/v on silica

EthylSO₂—D-Phe-Pro-Lys-(2-thiazolyl).

EthylSO₂—D-Phe-Pro-Lys(Cbz)-(2-thiazolyl) (336 mg) was treated withtrifluoroacetic acid/thioanisole 10/1 v/V (44 ml) for 4 h at roomtemperature. The reaction mixture was concentrated in vacuo and theresidue was dissolved in water. The aqueous phase was washed extensivelywith diethyl ether. The water layer was concentrated in vacuo,coevaporated with diluted hydrochloric acid and lyophilised from water.The crude product was charged onto a preparative HPLC Deltapack C18 RPcolumn using a gradient elution system of 20% A/65%B/15% C to 20% A/20%B/60% C over 40 minutes, at a flow rate of 50 ml/min). Yield: 160 mg ofEthylSO₂—D-Phe-Pro-Lys-(2-thiazolyl).

R_(t) (LC): 39.47 min. 20% A/80% B to 20% A/20% B/60% C in 40 minutes.

EXAMPLE 23

D-Hpl-Pro-Lysz(2-thiazolyl) (Hpl=3-hexahydrophenyl lactic acid)

H—D-Hpl-OMe

H—D-Cha-OH (1.0 g) was dissolved in a mixture of 1N hydrochloric acid(4.8 ml), water (19.4 ml) and acetic acid (9.7 ml). At 0° C. a solutionof sodium nitrite (3.4 g) in water (5.8 ml) was added slowly and themixture was stirred overnight at room temperature. Hydrochloric acid,37%, (4.8 ml) was subsequently added and the mixture was stirred for 15min at room temperature. The reaction mixture was evaporated and theresidue was dissolved in ether/acetone. After filtration, the solutionwas concentrated in vacuo and the crude material was stirred in methanol(25 ml) for 18 h. The pH was 1.5. The reaction mixture was evaporated todryness and the residue was purified by chromatography on silica(eluent: toluene/methanol 97/3 v/v. to yield H—D-Hpl-OMe (612 mg)

TLC: Rf=0.9, ethyl acetate/pyridine/acetic acid/water 163/20/6/11v/v/v/v on silica.

THP-D-Hpl-OMe (THP=tetrahydropyran)

To a stirred solution of H—D-Hpl-OMe(450 mg) in dichloromethane (2 ml)was successively added 3,4-dihydro-2H-pyran (0.285 ml) and pyridiniump-toluenesulfonate (60 mg). The mixture was stirred for 6 h. at roomtemperature and diluted with ether. This mixture was washed with brine,dried over sodium sulfate, filtered and evaporated in vacuo. The crudematerial was purified by chromatography on silica (eluent: ethylacetate/heptane 1/4 v/v) to yield THP-D-Hpl-OMe (498 mg).

TLC: Rf=0.64, ethyl acetate/heptane 1/2 v/v on silica.

THP-D-Hpl-OH

A solution of THP-D-Hpl-OMe (10.3 g) in of dioxane/water 9/1 (200 ml)was treated with sufficient 1N sodium hydroxide to keep the pH at 12 for18 hours at room temperature. After acidification, the mixture waspoured into water (500 ml) and extracted with dichloromethane. Theorganic layer was washed with water and dried on sodium sulfate Thefiltrate was evaporated and yielded 6.6 g of the title compound.

TLC: Rf=0.78, ethyl acetate/pyridine/acetic acid/water 163/20/6/11v/v/v/v on silica.

THP-D-Hpl-Pro-OH

To a solution of THP-D-Hpl-OH (5.87 g) in acetonitrile (75 ml). wassuccessively added EDCI (1-(3-dimethylaminopropyl)-3-ethylcarbodiimidehydrochloride) (4.84 g) and N-hydroxysuccinimide (2.9 g). The reactionmixture was stirred at room temperature for 16 h. The mixture wasconcentrated in vacuo and the residue was dissolved in ethyl acetate.This solution was washed with water and brine, dried over sodium sulfateand concentrated in vacuo. The crude material was dissolved indimethylformamide (100 ml) and added to a solution of proline.HCl (6.99g) in dimethylformamide/water, 1/1, v/v (200 ml), which was adjusted toa pH of 8.5 by sodium hydroxide. After stirring overnight the reactionmixture was concentrated in vacuo and the residue was dissolved inwater. This aqueous solution was adjusted to pH 2.5 at 0° C., followedby extraction with ethyl acetate. The combined organic layers weresuccessively washed with water and brine, dried over sodium sulfate andconcentrated in vacuo. The crude material was purified by chromatographyon silica (eluent: ethyl acetate/methanol, 8/2 6/4, v/v %) to yieldTHP-D-Hpl-Pro-OH (6.75 g).

TLC: Rf=0.52, ethyl acetate/pyridine/acetic acid/water 163/20/6/11v/v/v/v on silica.

THP-D-Hpl-Pro-Lys(Cbz)-(2-thiazolyl)

THP-D-Hpl-Pro-OH (390 mg) was dissolved in dry dimethylformamide (5 ml).After addition of ethyl diisopropyl amine (0.19 ml), the reactionmixture was placed under nitrogen and cooled to −20° C.Isobutylchloroformate (130 ml) was subsequently added and the mixturewas allowed to stir for 15 min at −20° C. H-Lys(Cbz)-(2-thiazolyl).TFA(1.05 eq.) was dissolved in dry dimethylformamide (5 ml) and addeddropwise to the cold mixed anhydride solution, maintaining the pH at 8.5by addition of ethyl diisopropyl amine. The reaction mixture was stirredfor 15 min at −20° C. and 2.5 h. at room temperature. The reactionmixture was evaporated to dryness. The residue was dissolved in ethylacetate and successively washed with 5% aqueous sodium hydrogencarbonatesolution, water and brine, dried over sodium sulfate and concentrated invacuo. The residue was purified by chromatography on silica (eluent:ethyl acetate/heptane 2/1 v/v) to yieldTHP-D-Hpl-Pro-Lys(Cbz)-(2-thiazolyl) (497 mg).

TLC: Rf=0.42 in ethyl acetate/heptane 2/1 v/v on silica.

D-Hpl-Pro-Lys-(2-thiazolyl)

THP-D-Hpl-Pro-Lys(Cbz)-(2-thiazolyl) (470 mg) was treated withtrifluoroacetic acid/thioanisole 10/1 v/v (38.5 ml) for 4 h at roomtemperature. The reaction mixture was concentrated in vacuo and theresidue was dissolved in water. The aqueous phase was washed extensivelywith diethyl ether. The water layer was concentrated in vacuo,coevaporated with diluted hydrochloric acid and lyophilised from water.The crude product was charged onto a preparative HPLC Deltapack C18 RPcolumn using a gradient elution system of 20% A/65%B/15% C to 20% A/20%B/60% C over 40 minutes, at a flow rate of 50 ml/min. Yield: 75 mg ofD-Hpl-Pro-Lys-(2-thiazolyl).

R_(t) (LC): 40.00 min. 20% A/80% B to 20% A/20% B/60% C in 40 minutes.

EXAMPLE 24 HOOC—CH₂—D-Phe-Pro-Lys-(2-thiazolyl) HLD-Phe-OMe.HCl

To cold (−20° C.) and dry methanol (1 l) was added dropwisethionylchloride (130 ml). H—D-Phe-OH.HCl (147.6 g) was added and thereaction mixture was heated under reflux for 30 min and then kept atroom temperature overnight. The mixture was concentrated in vacuo andcoevaporated with methanol (3 times). The residue was crystallized frommethano/diethyl ether yielding H—D-Phe-OMe.HCl as a white crystallinepowder (187.4 g). TLC: R_(f)=0.54, silica gel, n-butanolaceticacid/water 10/1/3 v/v.

N-(t-Butyloxycarbonylmethyl)-D-Phe-OMe

t-Butyl-bromo acetate (65 ml) was added to a stirred solutionH—D-Phe-OMe.HCl (65.2 g) in 400 ml of acetonitrile. The pH of themixture was adjusted to 8.5 with N,N-diisopropylethylamine. The mixturewas stirred for 16 hours at room temperature and evaporated in vacuo.The residue was dissolved in dichloromethane and the solution was washedwith water, dried on sodium sulfate and evaporated in vacuo.Chromatography over silica gel in heptane/ethyl acetate 9/1 (v/v) gave96.4 g of N-(t-butyloxycarbonylmethyl)-D-Phe-OMe. TLC: R_(f)=0.90,silica gel, ethyl acetate/pyridine/acetic acid/water 376/31/18/7v/v/v/v.

N-(t-Butyloxycarbonylmethyl)—N-Boc-D-Phe-OMe

The pH of a solution of N-(t-butyloxycarbonylmethyl)-D-Phe-OMe (96.4 g)and di-t-butyl dicarbonate (72.2 g) in N,N-dimethyl formamide (400 ml)was adjusted to 8.5 with N,N-diisopropylethylamine. The mixture wasstirred for 48 h at room temperature. The solvent was removed in vacuo.Dichloromethane and water were added to the residue. The organic layerwas separated, washed with cold 1N hydrogen chloride, water, saturatedsodium hydrogen carbonate solution and water. The organic layer wasdried over sodium sulfate and the filtrate was evaporated. The residuewas chromatographed on silica in toluene/ethyl acetate 9/1 (v/v) ascluent. The fractions containingN-(t-Butyloxycarbonylmethyl)-N-Boc-D-Phe-OMe were pooled and evaporated.Yield: 115.3 g. TLC: R_(f)=0.77, silica gel, toluene/ethyl acetate 9/1v/v.

N-(t-Butyloxycarbonylmethyl)—N-Boc-D-Phe-OH

A solution of N-(t-butyloxycarbonylmethyl)-N-Boc-D-Phe-OMe (115.3 g) in800 ml of dioxane/water=9/1 (ylv) was treated with sufficient 2N sodiumhydroxide to keep the pH at 12 for 16 hours at room temperature. Afteracidification, the mixture was poured into water stand was extractedwith dichloromethane. The organic layer was washed with water and wasdried over sodium sulfate. The filtrate was evaporated and yielded 104 gof N-(t-butyloxycarbonylmethyl)-N-Boc-D-Phe-OH.

TLC: R_(f)=0.10, silica gel, toluene/ethyl acetate 7/3 v/v.

N-(t-Butyloxycarbonylmethyl)-N-Boc-D-Phe-Pro-OBzl

To a cold (0° C.) solution ofN-(t-butyloxycarbonylmethyl)-N-Boc-D-Phe-OH (5.3 g) in N,N-dimethylformamide (40 ml) were successively added 1-hydroxy benzotriazole (2.8g), dicyclohexyl carbodiimide (3.2 g), H-Pro-OBzl.HCl (3.78 g) andtriethylamine (2.16 ml). The mixture was stirred at 0° C. for 1 hour andthen kept at room temperature overnight. The mixture was cooled to −20°C. and dicyclohexylurea was removed by filtration. The filtrate wasevaporated to dryness. The residue was dissolved in ethyl acetate andwashed successively with 5% sodium hydrogen carbonate, water, 2% citricacid and brine, dried over sodium sulfate and concentrated in vacuo. Theresidue was chromatographed on silica gel in heptane/ethyl acetate 6/4(v/v ) as eluent. The fractions containingN-(t-butyloxycarbonylmethyl)-N-Boc-D-Phe-Pro-OBzl were pooled andevaporated. Yield: 4.35 g. TLC: R_(f)=0.74, silica gel, heptane/ethylacetate 1/1 v/v.

N-(t-Butyloxycarbonylmethyl)-N-Boc-D-Phe-Pro-OH

10% palladium on charcoal (450 mg) was added to a solution ofN-(t-Butyloxy-carbonylmethyl)-N-Boc-D-Phe-Pro-OBzl (4.35 g) in methanol(50 ml). The mixture was hydrogenated at atmospheric pressure at roomtemperature for 45 min. The palladium catalyst was removed by filtrationand the solvent was removed by evaporation at reduced pressure yielding3.48 g N-(t-Butyloxycarbonylmethyl)-N-Boc-D-Phe-OH.

TLC: R_(f)=0.63, silica gel, ethyl acetate/pyridine/acetic acid/water664/31/18/7 v/v/v/v.

N-(t-Butyloxycarbonylmethyl)-N-Boc-D-Phe-Pro-Lys(Cbzh(2-thiazolyl)

To a cooled (−20° C.) solution of 375 mgN-(t-butyloxycarbonylmethyl)-N-Boc-D-Phe-Pro-OH and 276 mlN,N-diisopropylethylamine in 10 ml of N,N-dimethyl formamide, 100 mlisobutyl chloroformate was added. The reaction mixture was stirred foranother 20 minutes at −20° C. H-Lys(Cbz)-(2-thiazolyl).TFA (362 mg) wasdissolved in 5 ml N,N-dimethyl formamide and adjusted to pH 8 withN,N-diisopropylethylamine. This solution was slowly added to thereaction mixture. The reaction mixture was stirred for 15 min at −20° C.and then allowed to warm up to room temperature. The reaction mixturewas evaporated to dryness and the residue was dissolved in ethylacetate. The organic phase was washed with 5% sodium hydrogencarbonate,water and brine, dried over sodium sulfate and concentrated to give 622mg of crude product. Silica gel purification, usingdichloromethane/methanol 97/3 v/v as eluent, afforded 394 mg ofN-(t-butyloxycarbonylmethyl)-N-Boc-D-Phe-Pro-Lys(Cbz)-(2-thiazolyl).TLC: R_(f)=0.50, silica gel, dichloromethane/methanol 95/5 v/v.

HOOC—CH₂—D-Phe-Pro-Lys-(2-thiazolyl)

The protected tripeptide (394 mg) was treated with trifluoroacetic acidand thioanisole according to the procedures described in example 1 toafford, after HPLC purification, 206 mg ofHOOC—CH₂—D-Phe-Pro-Lys2-thiazolyl).

R_(t) (C); 27.9 min, 20% A, 80% B to 20a A, 20% B and 60% C in 40 min.

EXAMPLE 25 HOOC-CH₂—D-p-OCH₃-Phe-Pro-Lys-(2-thiazolyl)

HOOC—CH₂—D-p-OCH₃-Phe-Pro-Lys-(2-thiazolyl) was prepared in a similarmanner as described in example 24, starting from H—D-p-OCH₃-Phe-OH. HCl.Deprotection (see example 1) of 345 mgN-(t-butyloxycarbonylmethyl)-N-Boc-D-p-OCH₃-Phe-Pro-Lys(Cbz)-(2-thiazolyl)gave, after HPLC purification, 153 mg of the product.

R_(t) (LC): 28.9 min, 20% A, 80% B to 20% A, 20% B and 60% C in 40 min.

EXAMPLE 26 HOOC—CH₂—D/L-m-F-Phe-Pro-Lys-(2-thiazolyl)N-(t-Butyloxycarbonylmethyl)-N-Boc-D/L-m-F-Phe-OH

According to analogous procedures as described in example 24,H—DAL-m-F-Phe-OH. HCl (5 g) was converted intoN-(t-butyloxycarbonylmethyl)-N-Boc-D/L-m-F-Phe-OH. Yield: 8 g. TLC:R_(f)=0.65, silica gel, ethyl acetate/methanol 9/1 v/v.

N-(t-Butyloxycarbonylmethyl)-N-Boc-D/L-m-F-Phe-Pro-OMe

To a cold (0° C.) solution ofN-(t-butyloxycarbonylmethyl)-N-Boc-D/L-m-F-Phe-OH (7.9 g) inN,N-dimethyl formamide (80 ml) were successively added 1-hydroxybenzotriazole (4.0 g), dicyclohexyl carbodiimide (4.5 g), H-Pro-OMe.HCl(3.6 g) and triethylamine (3.25 ml). The mixture was stirred at 20° C.for 1 hour and then kept at room temperature overnight. The mixture wascooled to −20° C. and dicyclohexylurea was removed by filtration. Thefiltrate was evaporated to dryness. The residue was dissolved in ethylacetate and washed successively with 5% sodium hydrogencarbonate, water,2% citric acid and brine, dried over sodium sulfate and concentrated invacuo. The residue was purified over silica gel in heptane/ethyl acetate7/3 v/v, to afford 6.9 g of the product. TLC: R_(f)=0.65 heptane/ethylacetate 1/1, v/v.

N-(t-Butyloxycarbonylmethyl)-N-Boc-D/L-m-F-Phe-Pro-OH

6.9 g of N-(t-Butyloxycarbonylmethyl)-N-Boc-D/L-m-F-Phe-Pro-OMe,dissolved in dioxane/water: 9/1 v/v (60 ml), was treated with a 1 Nsodium hydroxide solution (13.8 ml) portionwise over 16 hours, keepingthe pH at 10-10.5. The reaction mixture was diluted with ice-water andacidified with 2 N hydrogen chloride solution until pH 2. The aqueouslayer was extracted with dichloromethane. Next, the organic phase waswashed with cold water, dried over sodium sulfate and concentrated togive 14.7 g crude material. Purification over silica gel in ethylacetate/methanol 9/1 v/v afforded 5.22 g. TLC: R_(f)=0.20, silica gel,ethyl acetate/methanol 8/2 v/v.

N-(t-Butyloxycarbonylmethyl)-N-Boc-D/L-m-F-Phe-Pro-Lys(Cbz)-(2-thiazolyl)

Coupling of N-(t-butyloxycarbonylmethyl)-N-Boc-D/L-m-F-Phe-OH (601.3 mg)with H-Lys(Cbz)-(2-thiazolyl) was performed under the same conditions asdescribed in example 24. Yield: 684.3 mg. TLC: R_(f)=0.74, silica gel,dichloromethane/methanol 95/5 v/v.

HOOC—CH₂—D/L-m-F-Phe-Pro-Lys-(2-thiazolyl)

N-(t-butyloxycarbonylmethyl)-N-Boc-D/L-m-F-Phe-Pro-Lys(Cbz)-(2-thiazolyl)(673.5 mg) was treated under the same conditions with trifluoroaceticacid and thioanisole as described in example 1 to afford 259 mg of pureproduct after HPLC-purification.

R_(t) (LC): 28.4 min and 29.0 min, 20% A, 80% B to 20% A, 20% B and 60%C in 40 min.

EXAMPLE 27 HOOC—CH₂—D-p-CF₃-Phe-Pro-Lys-(2-thiazolyl)N-(t-Butyloxycarbonylmethyl)N-Boc-D/L-p-CF₃-Phe-OH

According to analogous procedures as described in example 24,H—D/L-p-CF₃-Phe-OH. HCl (10.12 g) was converted intoN-(t-Butyloxycarbonylmethyl)-N-Boc-D/L-p-CF₃-Phe-OH. Yield: 12.23 g.TLC: R_(f)=0.64, silica gel, ethyl acetate/methanol 9/1, v/v.

N-(1-Butylo yaronylmetbyM)-N-Boc-D-p-CF₃-Phe-Pro-OBzl

An amount of 6.10 g N-(t-Butyloxycarbonylmethyl)-N-Boc-D/L-p-CF3-Phe-OHwas coupled to H-Pro-OBzl. HCl according to the same procedure asdescribed in example 24. After work-up, the diastereoisomers could beseparated by silica gel, using heptane/ethyl acetate 75/25 v/v, toafford 0.63 g of pureN-(t-Butyloxycarbonylmethyl)-N-Boc-D-p-CF₃-Phe-Pro-OBzl.

TLC: R_(f)=0.35, silica gel, heptane/ethyl acetate 7/3 ylv.

N-(t-butyloxocarbonylmethyl)-N-Boc-D-p-CF₃-Phe-Pro-Lys(Cbz)-(2-thiazolyl)

N-(t-butyloxycarbonylmethyl)-N-Boc-D-p-CF₃-Phe-Pro-OBzl (630 mg) wasreduced and subsequently coupled to H-Lys(Cbz)-(2-thiazolyl) using theprocedures as described in example 24. Yield: 317.7 mg. TLC: R_(f)=0.46,dichloromethane/methanol 95/5 v/v.

HOOC—CH₂—D-p-CF₃-Phe-Pro-Lys-(2-thiazolyl)

The removal of the protected groups fromN-(t-butyloxycarbonylmethyl)N-Boc-D-p-CF₃-Phe-Pro-Lys(Cbz)-(2-thiazolyl)(306.5 mg) was performed using the same method as described in example24. After HPLC purification 157 mg of product was isolated.

R_(t) (LC)=36.7 min, 20% A, 80% B to 20% A, 20% B and 60% C in 40 min.

EXAMPLE 28 HOOC—CH₂—D-p-Cl-Phe-Pro-Lys-(2-thiazolyl)N-(t-Butyloxycarbonylmethyl)-N-Boc-D-p-Cl-Phe-OH

According to analogous procedures as described in example 24,H—D-p-Cl-Phe-OH. HCl (10 g) was converted intoN-(t-Butyloxycarbonylmethyl)-N-Boc-D-p-Cl-Phe-OH. Yield: 16.7 g. TLC:R_(f)=0.27, silica gel, ethyl acetate/methanol 9/1, v/v.

N-(t-butyloxycarbonylmethyl)-N-Boc-D-p-Cl-Phe-ONSu

A solution of N-(t-butyloxycarbonylmethyl)-N-Boc-D-p-Cl-Phe-OH (14.67 g)in 250 ml acetonitrile was treated with N-hydroxysuccinimide (4.11 g)and 1-(3-dimethylaminopropyl)3-ethylcarbodiimide (EDCI) hydrochloride(6.86 g) overnight at room temperature. The reaction mixture wasevaporated to dryness and the residue was dissolved in ethyl acetate.The organic phase was washed with water, dried over sodium sulfate andconcentrated to afford 19.11 g active ester, which was directly used inthe next step.

N-(t-butyloxycarbonylmethyl)-N-Boc-D-p-Cl-Phe-Pro-OH

H-Pro-OH.HCl (10.79 g) was dissolved in 100 ml N,N-dimethyl formamideand 100 ml water. The pH of the reaction mixture was adjusted to 8 witha 1 N sodium hydroxide solution, whereafterN-(t-butyloxycarbonylmethyl)-N-Boc-D-p-Cl-Phe-ONSu (19.11 g), dissolvedin 120 ml of N,N-dimethyl formamide, was added dropwise. The reactionwas stirred overnight at room temperature at pH 8. The reaction mixturewas cooled and adjusted to pH 2 with 1 N hydrochloric acid. The aqueouslayer was extracted with dichloromethane. The organic phase was washedwith water, dried over sodium sulfate en evaporated in vacuo. Silica gelpurification, using a gradient ethyl acetate/methanol 9/1 TLC:R_(f)=0.24, silica gel, ethyl acetate/methanol 8/2 v/v.

N-(t-butyloxycarbonylmethyl)-N-Boc-D-p-Cl-Phe-Pro-Lys(Cbz)-(2-thiazolyl)

According to the procedures described in example 24,N-(t-butyloxycarbonylmethyl)-N-Boc-D-p-Cl-Phe-OH (369.4 mg) wasconverted into the target compound. Yield: 249.1 mg.

TLC: R_(f)=0.25, silica gel, dichloromethane/methanol 97/3 v/v.

HOOC—CH₂—D-p-Cl-Phe-Pro-Lys-(2-thiazolyl)

As described in example 1, 231.5 mgN-(t-butyloxycarbonylmethyl)-N-Boc-D-p-Cl-Phe-Pro-Lys(Cbz)(2-thiazolyl)was deprotected and purified to obtain 109.8 mg of the product.

R_(t) (LC)=33.8 min, 20% A, 80% B to 20% A, 20% B and 60% C in 40 min.

EXAMPLE 29 HOOC—CH₂—D-o-Cl-Phe-Pro-Lys-(2-thiazolyl)

HOOC—CH₂—D-o-Cl-Phe-Pro-Lys-(2-thiazolyl) was prepared in a similarmanner as described in example 26, starting from H—D/L-o-Cl-Phe-OH.HCl.The two diastereoisomers were separated in the protected tripeptidestage. Deprotection of 230 mg ofN-(t-butyloxycarbonylmethyl)N-Boc-D-o-Cl-Phe-Pro-Lys(Cbz)-(2-thiazolyl),according to the method described in example 1, afforded 116 mg productafter HPLC purification.

R_(t) (LC)=30.0 min, 20% A, 80% B to 20% A, 20% B and 60% C in 40 min.

EXAMPLE 30 HOOC—CH—D/L-m,p-di-F-Phe-Pro-Lys-(2-thiazolyl)

This compound was prepared in a similar manner as described in example26, starting from H—D/L-m,p-di-F-Phe-OH.HCl. Removal of the blockinggroups of the protected tripeptide (720 mg) followed by HPLCpurification, as described in example 1, afforded 170 mg of the product.

R_(t) (LC)=30.7 min and 31.1 min, 20% A, 8O% B to 20% A, 20% B and 60% Cin 40 min.

EXAMPLE 31 HOOC—CH₂—D/L-o,p-di-Cl-Phe-Pro-Lys-(2-thiazolyl)

This compound was prepared in a similar manner as described in example26, starting from H—D/L-o,p-di-aPhe-OH.HCl. Removal of the blockinggroups of the protected tripeptide (1.07 g) followed by HPLCpurification, as described in example 1, afforded 100 mg of the product.

R_(t) (LC)=35.4 min and 36.1 min, 20% A, 80% B to 20% A, 20% B and 60% Cin 40 min.

EXAMPLE 32 HOOC—CH₂—D-Tyr-Pro-Lys-(2-thiazolyl) Cbz-D-Tyr(tBu)-OH

N-benzyloxycarbonyloxysuccinimide (5.75 g) was added to a suspension ofD-Tyr(tBu)-OH (5.0 g) in N,N-dimethyl formamide (40 ml). The pH of thesolution was adjusted to 8 using triethylamine. The reaction mixture wasstirred overnight at room temperature and then evaporated to dryness invacuo. The residue was dissolved in dichloromethane and diluted with icewater. The pH of the water layer was adjusted to pH 2.5 with 2 Nhydrogen chloride. The organic layer was seperated and the aqueous phasewas extracted with dichloromethane. The organic layers were combined andwashed with water, dried over sodium sulfate and concentrated. Yield:9.95 g. TLC: Rf=0.31, heptane/ethyl acetate 1/1.

Cbz-D-Tyr(tBu)OMe

[2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate](7.45 g) was added to a solution of Cbz-D-Tyr(tBu)-OH (9.95 g) indichloromethane (45) and methanol (5 ml). The pH of the mixture wasadjusted to 8 with N,N-diisopropylethylamine. The reaction mixture wasstirrred for 1 hour at room temperature and then quenched with 5% sodiumhydrogencarbonate. The organic phase was separated and washed withwater, 2% citric acid and brine, dried over sodium sulfate andconcentrated under reduced pressure. Yield: 10.2 g.

TLC:Rf=0.74, heptane/ethyl acetate 1/1.

H—D-Tyr(tBu)-OMe.HCl

10% palladium on charcoal (1.2 g) was added to a solution ofCbz-D-Tyr(tBu)-OMe (10.2 g) in methanol (100 ml) and 4N hydrogenchloride (5 ml). The mixture was hydrogenated at atmospheric pressure atroom temperature for 2 hours. The palladium catalyst was removed byfiltration. The filtrate was concentrated to a small volume followed bycystallisation from diethyl ether. Yield: 5.87 g. TLC: Rf=0.10,heptane/ethyl acetate 1/1.

HOOC—CH₂—D-Tyr-Pro-Lys-(2-thiazolyl)

This compound was prepared in a similar manner as described in example24, starting from H—D-Tyr(tBu)-OMe.HCl. Deprotection of 586 mgN-(t-butyloxycarbonylmethyl)-N-Boc-D-Tyr(tBu)-Pro-Lys(Cbz)-(2-thiazolyl),according to the procedures described in example 1, gave 283 mg of theproduct, after HPLC purification.

R_(t) (LC)=20.9 min, 20% A, 80% B to 20% A, 20% B and 60% C in 40 min.

EXAMPLE 33 HOOC—CH₂—D/L-p-CH₃-Phe-Pro-Lys-(2-thiazolyl)H—D/L-p-CH₃-Phe-OH.HCl

A suspension of sodium hydride (3.28 g, 60% dispersion in mineral oil)in ethanol (40 ml) was added to a solution of a-chloro-p-xylene (10 g),diethyl acetamidomalonate (19.3 g) and sodium iodide (8.55 g) in dioxane(80 ml) and ethanol (20 ml). The reaction mixture was refluxed at 80° C.for 90 min. The solvent was removed under reduced pressure and theresidue was dissolved in ethyl acetate. The organic phase was washedwith 5% sodium hydrogensulfate, 5% sodium hydrogensulfite, water, 5%sodium hydrogencarbonate and brine, dried over sodium sulfate andconcentrated in vacuo. The product was crystallised from heptane toafford 19.8 g of the condensation product. This was treated with 6 Nhydrogen chloride (420 ml) and acetic acid (210 ml) overnight at 95° C.to afford, after evaporation to dryness, 21.6 g of the product.

TLC: Rf=0.15, silca gel, ethyl acetate/pyridine/acetic acid/water664/31/18/7 v/v/v/v .

HOOC—CH₂—D/L-p-CH₃-Phe-Pro-Lys-(2-thiazolyl)

According to the same methods as described in example 24,HOOC—CH₂—D/L-p-CH₃-Phe-Pro-Lys-(2-thiazolyl) was prepared starting fromH—D/L-p-CH₃-Phe-OH.HCl. Removal of the blocking groups from theprotected tripeptide (582 mg) and HPLC purification was performed undersimilar conditions as described in example 1. Yield: 120 mg.

R_(t) (LC)=31.9 min, 20% A, 80% B to 20% A, 20% B and 60% C in 40 min.

EXAMPLE 34 HOOC—CH₂—D-m-Cl-Phe-Pro-Lys-(2-thiazolyl)

Starting from 3-chloro benzylbromide, H—D/L-m-Cl-Phe-OH.HCl was preparedas described in example 33. Next, the fully protected tripeptide wasassembled according to the same procedures as described in example 26.In the final step 1 gN-(t-butyloxycarbonylmethyl)-N-Boc-D/L-m-Cl-Phe-Pro-Lys(Cbz)-(2-thiazolyl)was treated with trifluoroacetic acid and thioanisole (see example 1).After HPLC purification 195 mg HOOC—CH₂—D-m-Cl-Phe-Pro-Lys-(2-thiazolyl)was isolated.

R_(t) (LC)=31.7 min, 20% A, 80% B to 20% A, 20% B and 60% C in 40 min.

EXAMPLE 35 HOOC—CH₂—D-DPA-Pro-Lys-(2-thiazolyl). (DPA=diphenylalanine)

This compound was prepared in a similar manner as described in example24, starting from H—D-DPA-OH.HCl. Deprotection of 570 mgN-(t-butyloxycarbonylmethyl)-N-Boc-D-DPA-Pro-Lys(Cbz)-(2-thiazolyl),according to the methods described in example 1, afforded after HPLCpurification 194 mg end product.

R_(t) (LC)=35.6 min, 20% A, 80% B to 20% A, 20% B and 60% C in 40 min.

EXAMPLE 36 HOOC—CH₂—D-m-OH-Phe-Pro-Lys-(2-thiazolyl)

This compound was prepared in a similar manner as described in example24, starting from H—D/L-m-OH-Phe-OH.HCl. The phenolic hydroxyl functionwas also protected with a Boc group during the introduction of the Bocgroup on the N-terminus. Deprotection (see example 1) of 1.21 gN-(t-butyloxycarbonylmethyl)-N-Boc-D/L-m-Boc-Phe-Pro-Lys(Cbz)-2-thiazolyl)afforded after HPLC purification the desired diastereoisomer. Yield: 99mg.

R_(t) (LC)=23.8 min, 20% A, 80% B to 20% A, 20% B and 60% C in 40 min.

EXAMPLE 37 HOOC—Ch₂—D/L-m-OCH₃-Phe-Pro-Lys-(2-thiazolyl)Boc-D/L-mOH-Phe-OH

H—D/L-m-OH-Phe-OH.HCl (5.25 g) was dissolved in dioxane (55 ml), water(28 ml) and 1 N sodium hydroxide solution (29.0 ml). Di-t-butyldicarbonate (6.95 g) was added and the reaction mixture was stirredovernight at room temperature at pH 9. The reaction mixture was dilutedwith water (200 ml) and extracted with heptane. The aqueous layer wasdiluted with ethyl acetate (150 ml) and acidified to pH 2 with 1Nhydrogen chloride. The organic phase was separated and the water layerwas extracted with ethyl acetate. The organic layers were combined andwashed with water and brine, dried over sodium sulfate and concentratedin vacuo. Yield: 8.49 g.

TLC: R_(t)=0.67, silica gel, ethyl acetate/pyridine/acetic acid/water126/20/6/11 v/v/v/v.

Boc-D/L-m-OCH₃-Phe-OMe

A mixture of Boc-D/L-m-OH-Phe-OH (8.49 g), sodium carbonate (23.9 g) andiodomethane (20.3 ml) in N,N-dimethyl formamide (60 ml) was stirred at60° C. for 48 h. Next, the reaction mixture was poured into ice-waterand acidified to pH 2.5 with 2 N hydrogen chloride, followed byextraction with ethyl acetate. The organic layers were combined andwashed with water and brine, dried over sodium sulfate and concentratedin vacuo. The crude product was purified by silica gel chromatographyusing heptane/ethyl acetate 7/3 v/v. Yield: 6.66 g.

TLC: R_(t)=0.56, silica gel, heptane/ethyl acetate 3/2 v/v.

H—D/L-m-OCa-Phe-OMe.TFA

Boc-D/L-m-OCH₃-Phe-OMe (6.66 g) was dissolved in dichloromethane (20 ml)and trifluouroacetic acid (20 ml) and stirred at room temperature for 2hours. The solvent was removed under reduced pressure and the crudeproduct was coevaporated from toluene twice. Yield: 9.56 g.

TLC: R_(t)=0.32, silica gel, ethyl acetate/pyridine/acetic acid/water126/20/6/11 v/v/v/v.

HOOC—CH₂—D/L-m-OCH₃-Phe-Pro-Lys-(2-thiazolyl)

H—D/L-m-OCH₃-Phe-OMe.TFA was used to assembleN-(t-butyloxycarbonylmethyl)-N-Boc-D/L-m-OCH₃-Phe-Pro-Lys(Cbz)-(2-thiazolyl)according to the same route as described in example 24, Treatment of 624mg of the protected tripeptide with trifluoroacetic acid and thioanisole(see example 1), followed by HPLC purification afforded 114 mg of theproduct.

R_(t) (LC)=29.3 min and 29.8 min, 20% A, 80% B to 20% A, 20% B and 60% Cin 40 min.

EXAMPLE 38 HOOC—CH₂—D/L-p-Br-Phe-Pro-Lys-(2-thiazolyl)Boc-D/L-p-Br-Phe-OH

A suspension of H—D/L-p-Br-Phe-OH (2.44 g) in 25 ml t-butanol/water 1/1v/v was adjusted to pH 9 with sufficient diluted sodium hydroxide (1 N)solution. Di-t-butyl dicarbonate (3.27 g) was added and the reactionmixture was stirred overnight while the pH was kept at 9. The reactionmixture was diluted with water, followed by extraction with heptane. Thewater layer was diluted with ethyl acetate and subsequently acidified topH 2.5 using 2 N hydrogen chloride. The organic phase was separated andthe aqueous phase was extracted with ethyl acetate. The organic layerswere combined and washed with water and brine, dried over sodium sulfateand concentrated in vacuo. Yield: 3.35 g.

TLC: R_(t)=0.32, silica gel, ethyl acetate/pyridine/acetic acid/water126/20/6/11 v/v/v/v.

Boc-D/L-p-Br-Phe-Pro-OH

Boc-D/L-pBr-Phe-OH (3.35 g) was coupled with H-Pro-OMe.HCl andsubsequently saponified with sodium hydroxide according to the samemethods as described in example 26. Yield: 3.13 g. TLC: Rf=0.45, silicagel, ethyl acetate/methanol 9/1.

Boc-D/L-p-Br-Phe-Pro-Lys(Cbz)-(2-thiazolyl)

Coupling of Boc-D/L-p-Br-Phe-Pro-OH (750 mg) withH-Lys(Cbz)-(2-thiazolyl) was performed under the same conditions asdescribed in example 24. Yield: 1.01 g.

TLC: R_(f)=0.85, silica gel, dichloromethane/methanol 9/1 v/v.

H—D/L-p-Br-Phe Pr L s(Cbz42-thiazolyl.TFA

Boc-D/L-p-Br-Phe-Pro-Lys(Cbz)2-thiazolyl) (1.01 g) was dissolved intrifluoroacetic acid (TFA, 10 ml) and stirred for 1 hour at roomtemperature. The solvent was removed under reduced pressure. Yield: 879mg.

TLC: R_(f)=0.75 and 0.68, silica gel, ethyl acetate/pyridine/aceticacid/water 63/20/6/11 v/v/v/v.

N-(t-butyloxycarbonylmethyl)-D/L-p-Br-Phe-Pro-Lys(Cbz)-(2-thiazolyl)

t-Butyl bromoacetate (264 ml) was added to a solution ofH—D/L-p-Br-Phe-Pro-Lys(Cbz)-(2-thiazolyl).TFA (879 mg) in acetonitrile(25 ml). The pH of the reaction mixture was adjusted to 8 withN,N-diisopropylethylamine whereafter the reaction mixture was allowed tostand overnight at room temperature. The solvent was removed byevaporation and the residue was dissolved in ethyl acetate. The organicphase was washed with water, 5% sodium hydrogencarbonate and brine,dried over sodium sulfate and concentrated in vacuo. The crude productwas purified over silica gel using dichloromethane/methanol 95/5 v/v toafford 850 mg of the protected tripeptide.

TLC: R_(f)=0.91, silica gel, ethyl acetate/pyridine/acetic acid/water63/20/6/11 v/v/v/v.

HOOC—CH₂—D/L-p-Br-Phe-Pro-Lys-(2-thiazolyl)

N-(t-butyloxycarbonylmethyl)-D/L-p-Br-Phe-Pro-Lys(Cbz)-(2-thiazolyl)(850 mg) was treated under the same conditions with trifloroacetic acidand thioanisole as described in example 1 to obtain, after HPLCpurification, 123 mg of the product.

R_(t) (LC)=33.9 min and 34.4 min, 20% A, 80% B to 20% A, 20% B and 60% Cin 40 min.

EXAMPLE 39 HOOC—CH₂—D-p-F-Phe-Pro-Lys-(2-thiazolyl)

This compound was prepared in a similar manner as described in example38, starting from H—D-p-F-Phe-OH. Deprotection (see example 1) of 563 mgN-(t-butyloxycarbonylmethyl)-D-p-F-Phe-Pro-Lys(Cbz)-(2-thiazolyl) gave,after HPLC purification, 182 mg of the product.

R_(t) (LC)=29.7 min, 20% A, 80% B to 20% A, 20% B and 60% C in 40 min.

EXAMPLE 40 HOOC—CH₂—D/L-m,p-di-Cl-Phe-Pro-Lys-(2-thiazolyl)

This compound was prepared in a similar manner as described in example38, starting from H—D/L-m,p-di-Cl-Phe-OH. Deprotection (see example 1)of 480 mgN-(t-butyloxycarbonylmethyl)-D/L-m,p-di-Cl-Phe-Pro-Lys(Cbz)2-thiazolyl)gave, after HPLC purification, 191 mg of the product.

R_(t) (LC)=36.8 min and 37.8 min, 20% A, 8/B to 20% A, 20% B and 60% Cin 40 min.

EXAMPLE 41 BzlSO₂-norLeu(cyclo)Gly-LysΨ[COCO]—OH (Bzl=benzyl)Cbz-Lys(Boc)-OMe

Cbz-Lys(Boc)-OH (28 g) was dissolved in dichloromethane/methanol=9/1 v/v(500 ml). 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluroniumtetrafluoroborate (23.6 g) was added and the solution adjusted to pH 8by addition of triethylamine. The reaction mixture was stirred for 2 hat room temperature. The mixture was washed successively with cold 1Nhydrogen chloride solution, water, 5% sodium hydrogencarbonate, andwater and dried over sodium sulfate. The filtrate was evaporated and theresidue was chromatographed on silica gel in ethyl acetate/heptane=1/4v/v as eluent. The fractions containing Cbz-Lys(Boc)-OMe were pooled andevaporated. Yield: 29.1 g

TLC: R_(f)=0.85, silica gel, ethyl acetate/heptane=3/1 v/v.

Cbz-Lys(Boc)Ψ[cyanoacetate]

To a cold (−78° C.) solution of Cbz-Lys(Boc)-OMe (29.1 g) in drydichloromethane (800 ml) was added dropwise diisobutyl aluminumhydride(222 ml of 1M solution in hexane) at a rate to keep the reactiontemperature below −70° C. The resulting solution was stirred at −78° C.for 1 h. A 5% citric-acid solution (600 ml) was added to the reactionmixture. The two layer mixture was stirred at room temperature for 10minutes, the layers were separated and the aqueous layer was extractedwith dichloromethane. The combined dichloromethane layers were washedwith water and dried over sodium sulfate and filtered. The solution wasplaced under nitrogen and cooled on a icewater-bath. A solution ofsodium cyanide (36.3 g) and benzyltriethyl ammonium chloride (4.2 g) inwater (600 ml) was added. Under vigorous stirring acetic anhydride wasadded portionwise (2×9 ml) over a period of 30 min. The organic layerwas separated and the aqueous layer was extracted with dichloromethane.The combined dichloromethane layers were washed with water, dried oversodium sulfate, filtered and evaporated in vacuo. The residue waspurified by chromatography on silica (eluent heptane/ethyl acetate=1/1v/v) to yield Cbz-Lys (Boc)Ψ[cyanoacetate] (26.3 g.) .

TLC: Rf=0.60, silica gel, dichloromethane/ethyl acetate=7/3 v/V.

Cbz-Lys(Boc)Ψ[CHOHCO]—OMe

A solution of Cbz-Lys(Boc)Ψ[cyanoacetate] (26.3 g.) in diethylether/methanol=3/1 v/v (600 ml) was cooled to −20° C. under nitrogen,and 66 g of gaseous hydrochloric acid was introduced keeping thetemperature below −5° C. The reaction mixture was kept at 4° C.overnight. Water (100 ml) was added dropwise to the reaction mixturekeeping the temperature below 5° C. After stirring for 16 h at roomtemperature the organic layer was separated and washed with water. Theaqueous layer was saturated with sodium chloride and extracted withsec-butanoldichloromethane=3/2 v/v. The organic phase was washed withbrine, dried over sodium sulfate, filtered and evaporated in vacuo togive 25.4 g of the crude amine. The residue was taken up in N,N-dimethylformamide (400 ml), and bis-(tert-butyl)anhydride (16 g) was added andtriethylamine until pH 8. The reaction mixture was stirred at roomtemperature overnight. The solvent was removed by evaporation at reducedpressure. The residue was dissolved in ethyl acetate, washed with waterand brine successively, dried over sodium sulfate, filtered andevaporated in vacuo. The residue was purified by chromatography onsilica (eluent:

ethyl acetate/heptane=4/6 v/v) to yield Cbz-Lys(Boc)Ψ[CHOHCO]—OMe (15.8g).

TLC: Rf=0.75, silica gel, ethyl acetate/pyridine/aceticacid/water=63/20/6/11 v/v/v/v.

H-Lys(Boc)Ψ[CHOHCO]—OMe

10% palladium on charcoal (92 mg) and 2.18 ml of a 1N hydrochloridesolution were added to a solution of Cbz-Lys(Boc)Ψ[CHOHCO]—OMe (0.92 g)in N,N-Dimethyl formamide (20 ml). The mixture was hydrogenated atatmospheric pressure at room temperature for 3 h. The palladium catalystwas removed by filtration and the solvent was removed by evaporation atreduced pressure yielding H-Lys(Boc)Ψ[CHOHCO]—OMe.HCl quantitatively.

TLC: R_(f)=0.47, silica gel, ethyl acetate/pyridine/aceticacid/water=88/31/18/7 v/v/v/v.

BzlSO₂-norLeu(cyclo)Gly-Lys(Boc)Ψ[CHOHCO]—OMe

(S)-3-benzylsylfonylamido-2-oxo-1-azepineacetic acid was preparedaccording to the procedure in example 18. To a cold (0° C.) solution of(S)-3-benzylsulfonylamido-2-oxo-1-azepineacetic acid(BzlSO₂-norLeu(cyclo)Gly) (400 mg) in N,N-dimethyl formamide (20 ml)were successively added 1-hydroxy benzotriazole (238 mg), dicyclohexylcarbodiimide (267 mg), H-Lys(Boc)Ψ[CHOHCO]—OMe.HCl (385 mg) andtriethylamine (0.32 ml). The mixture was stirred at 0° C. for 1 hour andthen kept at room temperature overnight. The mixture was cooled to −20°C. and dicyclohexylurea was removed by filtration. The filtrate wasevaporated to dryness. The residue was dissolved in ethyl acetate andwashed successively with 5% sodium hydrogen carbonate, water, 2% citricacid, saturated aqueous sodium chloride, dried over sodium sulfate andconcentrated in vacuo. The residue was chromatographed on silica gel indichloromethane/methanol=9/1 (v/v) as eluent. The fractions containingBzlSO₂-norLeu(cyclo)Gly-Lys(Boc)Ψ[CHOHCO]—OMe were pooled andevaporated. Yield: 663 mg.

TLC: R_(f)=0.91, silica gel, ethyl acetate/pyridine/aceticacid/water=63/20/6/11 v/v/v/v.

BzlSO₂-norLeu(cyclo)Gly-Lys(Boc)Ψ[CHOHCO]—OH

BzlSO₂-norLeu(cyclo)Gly-Lys(Boc)Ψ[CHOHCO]—OMe (650 mg) was dissolved indioxane/water-7/3 v/v (20 ml) and treated with 2M sodium hydroxidesolution (1.05 ml) portionwise over 1 h at room temperature, keeping thepH at 12-13. The reaction mire was diluted with water (20 ml), 2Mhydrogen chloride solution was added until pH 2.0 and the water layerwas extracted with dichloromethane. The combined organic phases werewashed with water, brine and dried over sodium sulfate, filtered andconcentrated in vacuo to yieldBzlSO₂-norLeu(cyclo)Gly-Lys(Boc)Ψ[CHOHCO]—OH (740 mg).

TLC: R_(f)=0.44, silica gel, ethyl acetate/pyridine/aceticacid/water=63/20/6/11 v/v/v/v.

BzlSO₂-norLeu(cyclo)Gly-Lys(Boc)Ψ[COCO]—OH

To a solution of BnSO₂-norLeu(cyclo)Gly-Lys(Boc)Ψ[CHOHCO]—OH (740 mg) indry dichloromethane (20 ml) was added 450 mg of periodinane (Dess-Martinreagent). After 1 h stirring at room temperature, 2% sodium thiosulfatesolution was added (20 ml) and the mixture was stirred for 30 min atroom temperature. The organic layer was separated, washed with water,dried over sodium sulfate, filtered and evaporated in vacuo to givecrude BzlSO₂-norLeu(cyclo)Gly-Lys(Boc)Ψ[COCO]—OH (497 mg).

TLC: R_(f)=0.45, silica gel, ethyl acetate/pyridine/aceticacid/water=63/20/6/11 v/v/v/v.

BzlSO₂-norLeu(cyclo)Gly-LysΨ[COCO]—OH

BzlSO₂-norLeu(cyclo)Gly-Lys(Boc)Ψ[COCO]—OH (497 mg, crude) was treatedwith 90% trifluoroacetic acid/water (10 ml) for 1 h at room temperature.The reaction mixture was concentrated in vacuo and the residue dissolvedin water and directly charged onto a preparative HPLC DeltaPak RP-C₁₈using a gradient elution system of 20% A/80% B to 20% A/45% B/35% C over45 min at a flow rate of 80 ml/min. Yield: 200 mg ofBzlSO₂-norLeu(cyclo)Gly-LysΨ[COCO]—OH.

R_(t) (LC): 26.37 min; 20% A/80% B to 20% A/20% B/60% C in 40 min.

EXAMPLE 42 H—(—CH₃)—D-norLeu-Pro-LysΨ[COCO]—OH Boc-(N—CH₃)-NorLeu-Pro-OH

This compound was prepared according to example 11. In a similar manneras described in example 1 was prepared:H—(N—CH₃)—D-NorLeu-Pro-LysΨ[COCO]—OH Yield: 69 mg

R_(t) (LC): 13.27 min; 20% A/80% B to 20% A/20% B/60% C in 40 min.

EXAMPLE 43 H—D-Phe-Pro-LysΨ[COCO]—OH Boc-D-Phe-Pro-OMe

To a cold (0° C.) solution of Boc-D-Phe-OH (5 g) in N,N-dimethylformamide (200 ml) were successively added 1-hydroxy benzotriazole (4.29g), dicyclohexyl carbodimide (4.29 g), H-Pro-OMe.HCl (3.1 g) andN-ethylmorpholine (3 ml). The mixture was stirred at 0° C. for 1 hourand then kept at room temperature for 2 days. The mixture was cooled to−20° C. and dicyclohexylurea was removed by filtration. The filtrate wasevaporated to dryness. The residue was dissolved in ethyl acetate andwashed successively with 5% sodium hydrogen carbonate, 0.1M hydrogenchloride-solution, saturated aqueous sodium chloride, dried over sodiumsulfate and concentrated in vacuo. The residue was chromatographed onsilica gel in heptane/ethyl acetate=6/4 (v/v) as eluent. The fractionscontaining Boc-D-Phe-Pro-OMe were pooled and evaporated. Yield: 1.5 g.

TLC: R_(f)=0.90, silica gel, ethyl acetate/pyridine/aceticacid/water-163/20/6/11 v/v/v/v.

Boc-D-Phe-Pro-OH

Boc-D-Phe-Pro-OMe (8.3 g) was dissolved in dioxane/water=6/4 v/v (150ml) and treated with 2M sodium hydroxide solution (16.5 ml) portionwiseover 1 h at room temperature, keeping the pH at 12.5. 2M hydrogenchloride solution was added to the reaction mixture until pH 3.0 and thewater layer was extracted with ethyl acetate. The combined organicphases were washed with water, brine and dried over sodium sulfate,filtered and concentrated in vacuo to yield Boc-D-Phe-Pro-OH (6.9 g).

TLC: R_(f)=0.30, silica gel, ethyl acetate/pyridine/aceticacid/water=213/20/6/11 v/v/v/v.

In a similar manner as described in example 1 was prepared:H—D-Phe-Pro-LysΨ[COCO]—OH. Yield: 417 mg

R_(t) (LC): 16.22 min; 20% A/80%o B to 20% A/20% B/60% C in 40 min.

EXAMPLE 44 H—(N—CH₃)—D-Phe-(N-cyclopentyl)-Gly-LysΨ[COCO]—OHBoc-(N—CH₃)—D-Phe-(N-cyclopentyl)-Gly-OH

This compound was prepared as described in example 3 usingBoc-(N—CH₃)—D-Phe-OH and HCl.H-(N-cyclopentyl)-Gly-OMe.

TLC: R_(f)=0.52, silica gel, dichloromethane/methanol=9/1 v/v.

In a similar manner as described in example 1 was prepared:

H—(N—CH₃)—D-Phe-(N-cyclopentyl)-Gly-LysΨ[COCO]—OH. Yield: 87 mg

R_(t) (LC): 23.92 min; 20% A/80% B to 20% A/20% B/60% C in 40 min.

EXAMPLE 45 Ethylsulfonyl-D-Phe-Pro-LysΨ[COCO]—OHEthylsulfonyl-D-Phe-Pro-OH

This compound was prepared according to example 22.

In a similar manner as described in example 41 was prepared:

Ethylsulfonyl-D-Phe-Pro-LysΨ[COCO]—OH. Yield: 90 mg.

R_(t) (LC) 28.04 min; 20% A/80% B to 20% A/20% B/60% C in 40 mnm.

EXAMPLE 46(4aR,8aR)-perhydroisoquinoline-1(R)-carbonyl-Pro-LysΨ[COCO]—OH2-Cbz-(4aR,8aR)-perhydroisoquinoline-1(R)-carbonyl-Pro-OH

This compound was prepared according to example 20.

In a similar manner as described in example 41 was prepared:

(4aR,8aR)-perhydroisoquinoline-1(R)-carbonyl-Pro-LysΨ[COCO]—OH. Yield:170 mg.

R_(t) (LC): 18.95 min; 20% A/80% B to 20% A/20% B/60% C in 40 min.

EXAMPLE 47 HOOC—CH₂—D-Coa-Pro-Lys-(2-thiazolyl) (Coa=cyclo-octylalanine)Cyclo-octylmethyl Bromide

Cyclooctylmethanol (8.16 g) was dissolved in 47% HBr-solution (70 ml)and refluxed for 1 hour at 130° C. The reaction mixture was poured ontoicewater (500 ml) and saturated sodium hydrogencarbonate solution (500ml) was added. The aqueous solution was extracted with dichloromethane.The combined organic phases were washed with water, brine and dried oversodium sulfate, filtered and concentrated in vacuo. The residue waschromatographed on silica gel in toluene as eluent. The fractionscontaining cyclo-octylmethyl bromide were pooled and evaporated. Yield:9.85 g. TLC: R_(f)=0.95, silica gel, toluene

(R,S)-Ethyl-2acetylamino-2-cyano-3-cyclooctyl-propionate

Potassium tert.-butylate (6.85 g) and ethyl acetamidocyanoacetate (8.1g) were dissolved in dimethylsulfoxide (100 ml) at room temperature.Cycol-octylmethyl bromide was dissolved in dimethylsulfoxide (25 ml) andadded dropwise to the reaction mixture. The mixture was stirred at roomtemperature for 44 hours. After pouring onto 500 ml water theprecipitate was filtered and dried to yield(R,S)-ethyl-2-acetylamino-2-cyano-3-cyclooctyl-propionate (2.95 g)

TLC: R_(f)=0.50, silica gel, heptane/ethyl acetate=3/7 v/v.

H—D,L—Cyclo-octylalanine-OH.HCl

(R,S)ethyl-2-acetylamino-2-cyano-3-cyclooctyl-propionate (2.95 g) wassuspended in 100 ml of a 20%/hydrogen chloride-solution and refluxed for22 hours. The reaction mixture was cooled to 5° C. and the precipitateformed was filtered, washed with diethyl ether and dried.

Yield: 2.69 g H-D,L-Cyclo-octylalanine-OH.HCl (H—D,L—Coa-OH.HCl)

TLC: R_(f)=0.27, silica gel, ethyl acetate/pyridine/aceticacid/water=63/20/6/11 v/v/v/v.

In a similar manner as described in example 24 was prepared:HOOC—CH₂—D-Coa-Pro-Lys-(2-thiazolyl). Yield: 162 mg.

R_(t) (LC): 38.35 min; 20% A/80% B to 20% A/20% B/60% C in 40 min.

In a similar manner as described in example 24 were prepared:

EXAMPLE 48 HOOC—CH₂—D-2-Nal-Pro-Lys-(2-thiazolyl) (Nal=naphthylalanine)

Yield: 423 mg

R_(t) (LC): 35.78 min; 20% A/80% B to 20% A/20% B/60% C in 40 min

EXAMPLE 49 HOOC—CH₂—D-norLeu-Pro-Lys-(2-thiazolyl)

Yield: 344 mg

R_(t) (LC): 24.84 min; 20% A/80% B to 20% A/20% B/60% C in 40 min

EXAMPLE 50 HOOC—CH₂—D-Leu-Pro-Lys-(2-thiazolyl)

Yield: 138 mg

R_(t) (LC): 24.50 min; 20% A/80% B to 20% A/20% B/60% C in 40 min

EXAMPLE 51

Anti-thrombin Assay

Thrombin (Factor IIa) is a factor in the coagulation cascade.

The anti-thrombin activity of compounds of the present invention wasassessed by measuring spectrophotometrically the rate of hydrolysis ofthe chromogenic substrate s-2238 exterted by thrombin. This assay foranti-thrombin activity in a buffer system was used to assess theIC₅₀-value of a test compound.

Test medium: Tromethamine-NaCl-polyethylene glycol 6000 (TNP) buffer.Reference compound: I2581 (Kabi) Vehicle:TNP buffer. Solubilisation canbe assisted with dimethylsulphoxide, methanol, ethanol, acetonitrile ortert.-butyl alcohol which are without adverse effects in concentrationsup to 2.5% in the final reaction mixture.

Technique Reagents*: 1. Tromethamine-NaCl (TN) buffer. Composition ofthe buffer: Tromethamine(Tris) 6.057 g (50 mmol), NaCl 5.844 g (100mmol), water to 1 l. The pH of the solution is adjusted to 7.4 at 37° C.with HCl (10 mmol·l⁻¹). 2. TNP buffer: Polyethylene glycol 6000 isdissolved in TN buffer to give a concentration of 3 g·l⁻¹. 3. S-2238solution: One vial S-2238 (25 mg; Kabi Diagnostica, Sweden) is dissolvedin 20 ml TN buffer to give a concentration of 1.25 mg·ml⁻¹ (2 mmol·l⁻¹).4. Thrombin solution: Human thrombin (16 000 nKat·vial⁻¹; CentraalLaboratorium voor Bloedtransfusie, Amsterdam, The Netherlands) isdissolved in TNP buffer to give a stock solution of 835 nKat·ml⁻¹.Immediately before use this solution is diluted with TNP buffer to givea concentration of 3.34 nKat·ml⁻¹.

* All ingredients used are of analytical grade

For aqueous solutions ultrapure water (Milli-Q quality) is used.

Preparation of Test and Reference Compound Solutions

The test and reference compounds are dissolved in Milli-Q water to givestock concentrations of 10⁻² mol·l⁻¹. Each concentration is stepwisediluted with the vehicle to give concentrations of 10⁻³, 10⁻⁴ and 10⁻⁵mol·l⁻¹. The dilutions, including the stock solution, are used in theassay (final concentrations in the reaction mixture: 3·10⁻³; 10⁻³;3·10⁻⁴; 10⁻⁴; 3·10⁻⁵; 10⁻⁵; 3·10⁻⁶ and 10⁻⁶ mol·l⁻¹, respectively).

Procedure

At room temperature 0.075 ml and 0.025 ml test compound or referencecompound solutions or vehicle are alternately pipetted into the wells ofa microtiter plate and these solutions are diluted with 0.115 ml and0.0165 ml TNP buffer, respectively. An aliquot of 0.030 ml S-2238solution is added to each well and the plate is pre-heated andpre-incubated with shaking in an incubator (Amersham) for 10 min. at 37°C. Following pre-incubation the hydrolysis of S-2238 is started byaddition of 0.030 ml thrombin solution to each well. The plate isincubated (with shaking for 30 s) at 37° C. Starting after 1 min ofincubation, the absorbance of each sample at 405 nm is measured every 2min. for a period of 90 min. using a kinetic microtiter plate reader(Twinreader plus, Flow Laboratories).

All data are collected in an IBM personal computer using LOTUS-MEASURE.For each compound concentration (expressed in mol·l⁻¹ reaction mixture)and for the blank the absorbance is plotted versus the reaction time inmin.

Evaluation of responses: For each final concentration the maximumabsorbance was calculated from the assay plot. The IC₅₀-value (finalconcentration, expressed in ∥mol·l⁻¹, causing 50% inhibition of themaximum absorbance of the blank) was calculated using the logittransformation analysis according to Hafner et al.(Arzneim.-Forsch./Drug Res. 1977; 27(II): 1871-3).

In the following table, IC₅₀-values of compounds of the invention arelisted:

Example IC₅₀-value (μM) 2(e) 4.5 4(b) 4.34 39 19 41 0.135

We claim:
 1. A non-slow-binding thrombin inhibitor of the formula:A—B—C-Lys-D wherein A is —CO—(CH₂)_(n)COOH or —(CHR₂)_(n)COOH, wherein nis 1 or 2 and R₂ is H or methyl; B is selected from D-Cha, D-Coa,norLeu, Leu, Nal, DPA, Tyr and D-Phe optionally mono- or di-substitutedwith methoxy or halogen: C is Pro, or —N(cyclopentyl)-CH₂—CO—; D isthiazolyl or oxazolyl; or a prodrug thereof; or a pharmaceuticallyacceptable salt thereof.
 2. The non-slow-binding thrombin inhibitor ofclaim 1, wherein D is thiazolyl.
 3. A compound of the formula:HOOC—CH₂—D-Cha-Pro-Lys-(2-thiazolyl).
 4. A pharmaceutical compositioncomprising the non-slow-binding thrombin inhibitor of claim 1 or 2 and apharmaceutically acceptable auxiliary.
 5. A pharmaceutical compositioncomprising the compound of claim 3 and a pharmaceutically acceptableauxiliary.
 6. A process for preparing a non-slow-binding thrombininhibitor of claim 1, 2 or 3, comprising coupling suitably protectedamino acids or amino acid analogs, followed by removing the protectinggroups.
 7. A method for treating conditions involving undesired bloodcoagulation in a patient, comprising administering a thrombin-inhibitingeffective amount of a non-slow-binding thrombin inhibitor of any one ofclaim 1, 2 or 3 to said patient.
 8. A process for making apharmaceutical composition, comprising admixing a non-slow-bindingthrombin inhibitor of any one of claims 1, 2 or 3 with pharmaceuticallyacceptable auxiliaries.